The Role of Heat Shock Protein 70, IgE and MMP-9 in Detecting Early Minor Myocardial Damage and Evaluating the Efficacy of Coronary Artery Bypass Grafting (CABG)
The Role of Heat Shock Protein 70, IgE and MMP-9 in Detecting Early Minor Myocardial Damage and Evaluating the Efficacy of Coronary Artery Bypass Grafting (CABG)
Amal A. Baalash 1 , Hala E. Hamouda 1 , Ghada M. Ismail 2 , Ibrahim K. Yassein 3 and Bedir M. Ibrahim 3 1. Department of Medical Biochemistry, Faculty of Medicine, Tanta University, Tanta 31111, Egypt
2. Department of Physiology, Faculty of Medicine, Tanta University, Tanta 31111, Egypt 3. Department of Cardiothoracic Surgery, Faculty of Medicine, Tanta University, Tanta 31111, Egypt
Received: October 10, 2011 / Accepted: December 12, 2011 / Published: March 30, 2012.
Abstract: The objective of this research was to identify levels of heat shock protein 70 (Hsp 70), total immunoglobulin E (IgE) and matrix metalloproteinase-9 (MMP-9) before and after coronary artery bypass grafting (CABG) surgery. Hsp 70, IgE, MMP-9, creatine phosphokinase-MB (CPK-MB), and lactate dehydrogenase (LDH) levels were measured in normal subjects (n = 20), and in patients with chronic stable angina pectoris who were referred for elective CABG, before and after performing CABG-surgery (n = 20). Compared with normal subjects, increased Hsp 70 and IgE levels, unchanged MMP-9 level, and activities of CPK-MB and LDH were found in the pre-operative patient group. Hsp 70 and IgE levels in the post-operative period were significantly reduced when compared to pre-operative period. Hsp 70 and IgE might be used as markers for detection of early minor myocardial damage, and coronary insufficiency with less overt damage than myocardial infarction, as significant changes in their levels appear before occurrence of in any changes in the levels of MMP-9, CPK-MB and LDH. Besides, Hsp 70, and IgE returning to the normal levels after CABG surgery, suggest that they could be helpful to evaluate the effect of CABG surgery.
Key words: Heat shock protein, IgE, metaloproteinases, MMP-9, myocardial ischemia, coronary artery bypasses grafting.
1. Introduction such as C reactive protein (CRP), serum amyloid A protein, interleukin-6, TNF and interleukin-1 receptor
The clinical feature of myocardial ischemia antagonist, are commonly found in acute coronary correlates with a particular biochemical pattern of syndrome (ACS). It has been suggested that this inflammatory system activation. Experimental models systemic inflammatory response may be the result of of ischemic myocardial injury indicate that the the myocardial microinfarction known to occur in that inflammatory response after the ischemic event
case [2].
contributes to tissue damage [1]. So, inflammation is Heat shock proteins (Hsps) are abundant becoming an intriguing focus of research as a possible intracellular proteins found in both prokaryotic and pathogenetic component and therapeutic target in eukaryotic organisms. Their main function appears to ischemic heart disease.
be as chaperones, involved in protein folding and Elevated values of circulating inflammatory markers, transport [3]. Upregulation of the synthesis of a number
Corresponding author: Amal A. Baalash, Ph.D., MD, of these proteins occurs upon environmental stress that associate professor, research fields: medical biochemistry and
stablishes a unique defense system to maintain cellular biomarkers. E-mail: [email protected].
The Role of Heat Shock Protein 70, IgE and MMP-9 in Detecting Early Minor Myocardial Damage
and Evaluating the Efficacy of Coronary Artery Bypass Grafting (CABG)
protein homeostasis and to ensure survival of the cell. by reacting with the thiol group of the cysteine switch In the cardiovascular system, this enhanced protein
and forming an S-nitrosylated derivative, a demonstration synthesis leads a powerful increase in tolerance to such
of the chemical activation of a proMMP in vivo. endangering situations as ischemia, hypoxia, oxidative
MMP-9 is considered to play a role in the regulation injury, and endotoxemia. The Hsp 70 family, located in
of migration and proliferation of vscular smooth the cytosol and the nucleus of the cell, is the most
muscle cells (VSMCs) and modulates the cell-to-cell conserved and the best-studied class of Hsps. communication with activated surrounding cells, such Myocardial ischemia induces Hsp 70 as a stress
as inflammatory cells, endothelial cells and VSMCs response [4], and Hsp 70 is increased in atrial tissue of
patients with angina [5]. Circulating Hsp 70 is The activity of MMPs is normally low in healthy suggested as a marker of myocardial damage. In
tissue, but the expression and activity of several MMPs addition, Hsp 70 may has a role in the inflammatory
are increased in a range of pathological processes, such response after myocardial tissue damage [6].
as inflammation and tissue repair after ischemic Immunoglobulin E (IgE), known primarily as a
damage [10]. It has been reported that concentrations of mediator of allergy, can cause platelet activation and
MMPs are elevated not only in affected tissue and body arterial smooth muscle hyperplasia [7]. On the other
fluid, but also in peripheral blood in some patients with hand, it has been observed that myocardial tissue
cancer, liver cirrhosis or rheumatoid arthritis [14, 15]. damage is associated with an immunological response
These findings raise the possibility that patients with characterized by rise of serum IgE. This behavior of
ischemic myocardial tissue would show elevated serum IgE bears much resemblance to that of acute
peripheral blood levels of MMPs. phase proteins [8].
Early and accurate identification of myocardial Matrix metalloproteinases (MMPs) are a group of
damage in patients experiencing symptoms and signs endopeptidases with capacity to cleave components of
of coronary insufficiency is an important challenge. extracellular matrix, such as collagen and elastin. The
Current diagnostic tests for acute myocardial infarction ability to modify the tissues is important for several
(AMI) by conventional criteria are quite adequate. aspects of normal and abnormal physiology. Evaluation of new and more-sensitive tests that can Approximately 20 different MMPs are identified, and
detect early minor myocardial damage possess a new they can be subdivided into different groups according
problem.
to components of the extracellular matrix which they Cardiac markers of “minor myocardial damages” in degrade. Gelatinase B (MMP-9) belongs to the group
patients with stable angina pectoris (SAP) are not of gelatinases. It readily digests the denatured
confirmed and a marker of coronary insufficiency is collagens and gelatins. This group of enzymes has
not well established. Besides, the recovery markers three repeats of a type II fibronectin domain inserted in
after CABG surgery are also not well established. the catalytic domain, which bind to gelatin, collagens,
The present work aimed to study whether Hsp 70, and laminin [9].
total IgE, and MMP-9 which are proteins sharing in the MMPs are secreted in a latent proform and require
inflammatory response after myocardial tissue damage activation for proteolytic activity [10]. MMPs can be
and in tissue recovery, could be used as possible activated by proteinase, and reactive oxygen. Low pH
indicators for minor myocardial damages, and also to and heat treatment can also lead to activation [11].
study whether they could be used as markers for the Recently, studies by Gu et al. [12] have shown that
success of CABG surgery in improving the myocardial (NO) activates pro-MMP-9 during cerebral ischemia
ischemia.
The Role of Heat Shock Protein 70, IgE and MMP-9 in Detecting Early Minor Myocardial Damage and Evaluating the Efficacy of Coronary Artery Bypass Grafting (CABG)
2. Materials and Methods
age and sex were considered as the control group. All persons gave an informed consent for participation in
Twenty patients with chronic stable angina referred
the study.
for elective CABG were included in the study. The Blood samples were withdrawn from subjects after patients had angiographically verified three-vessel or overnight fasting and serum was separated for in a few cases two-vessel disease, were < 75 years old, determination of the following parameters: and before surgery they had no signs of infection. Also, (1) CPK-MB, and LDH using the commercially emergency cases and patients undergoing combined
available kits;
procedures or re-operations were excluded. (2) Serum levels of Hsp 70 was determined using The other exclusion criteria in the present study Hsp70 ELISA kits (StressGen Biotechnologies Corp); included other known diagnosed disease, acute phase (3) IgE levels were determined by solid phase fever, any allergic disease, oesinophilia or evidence of chemiluminescent immunometric assay, using immulite parasitic infestation in stool examination, and total IgE kits with IMMULITE 1000 analyzer; complications in the post-operative course. (4) Serum matrix metalloproteinase-9 (MMP-9) was
2.1 Anesthesia and Surgical Procedure determined using Ray Bio human MMP-9 ELISA kit (Ray Biotech. In).
All patients underwent a routine procedure with Data were expressed as means ± standard deviation median sternotomy and use of CPB. Anesthesia was (SD), statistical analysis of the result was performed induced with diazepam, fentanyl, thiopental, and using the student t-test for comparison between the pancuronium and maintained with isoflurane, fentanyl, different groups. In all cases, a difference at P < 0.05 and nitrous oxide until the start of CPB. The activated was considered statistically significant. clotting time was maintained > 480 seconds during
CPB, and CPB was performed with flow of 2.4 L/min
3. Results
per m 2 body surface area. Patients were cooled down to
34 °C at the beginning of CPB, and active rewarming The demographic data and the clinical was started during the last distal coronary anastomosis.
characteristics of the 20 patients who underwent the Antegrade intermittent warm blood cardioplegia was
elective CABG surgery were shown in Table 1. used. The left internal mammary artery was
The results of the current study revealed a significant anastomosed on the left anterior descending artery, and
difference in the serum levels of Hsp 70 between the venous grafts were used for the rest, with a mean of 2.8
pre-operative and post-operative samples of the same ± 0.8 grafts per patient.
patient (895 ± 123, and 311 ± 76 pg/mL, P < 0.05) Transit-time flowmetry was done for all patients
respectively, and the pre-operative samples and the intraoperatively to ensure graft patency and patients
samples taken from the control persons (895 ± 123 and who showed quantitative or qualitative low flow were
286 ± 112 pg/mL, P < 0.05) respectively. However excluded.
there was no statistically significant difference in its levels between both the control groups and the
2.2 Blood Samples
post-operative samples (Table 2).
Two blood samples were taken from the patients, the Table 3 shows that, total IgE levels were first one was taken at the day just before the surgery
significantly higher in the pre-operative samples (99.8 and the second one was taken three weeks after CABG
± 8.6 IU/mL) when compared with both the post- performance.
operative samples (37.4 ± 5.7 IU/mL) and the control Another 20 healthy normal persons with matched
ones (31.6 ± 3.4 IU/mL), and the post-operative samples
The Role of Heat Shock Protein 70, IgE and MMP-9 in Detecting Early Minor Myocardial Damage
and Evaluating the Efficacy of Coronary Artery Bypass Grafting (CABG)
didn’t show a statistically significant difference when
groups (Tables 4-6).
compared with the control samples.
4. Discussion
MMP-9, CPK, and LDH levels didn’t show anystatistically significant difference between the three
The most important finding in the present study is
Table 1 Demographic data and clinical characteristics of the patients undergoing elective CABG.
Characteristic Value Range Age, years
43-66
Sex, male/female
CPB time, min
35-95
Aortic cross-clamping, min
20-67
Intubation time, hour
4.0-8.1
Postoperative in-intensive care unit stay, day
Postoperative in-hospital stay, day
6-15
Distal coronary anastomoses per patient
2-4
Table 2 Serum levels of Hsp 70 (pg/mL) in all studied groups.
Control Pre-operative Post-operative Mean ± SD
P (compared to control)
P < 0.05*
P > 0.05
P (compared to postoperative group)
Table 3 Serum levels of Ige (IU/mL) in all studied groups.
Control Pre-operative Post-operative
Mean ± SD
P (compared to control)
P < 0.05*
P > 0.05
P (compared to postoperative group)
Table 4 Serum levels of MMP-9 (ng/mL) in all studied groups.
Control Pre-operative Post-operative
Mean ± SD
P (compared to control)
P > 0.05
P > 0.05
P (compared to postoperative group)
P > 0.05
P > 0.05
Table 5 Serum levels of CPK-MB (IU/L) in all studied groups.
Control Pre-operative Post-operative
Mean ± SD
P (compared to control)
P > 0.05
P > 0.05
P (compared to postoperative group)
P > 0.05
P > 0.05
Table 6 Serum levels of LDH (IU/L) in all studied groups.
Control Pre-operative Post-operative Mean ± SD
P (compared to control)
P > 0.05
P > 0.05
P (compared to postoperative G)
P > 0.05
P > 0.05
The Role of Heat Shock Protein 70, IgE and MMP-9 in Detecting Early Minor Myocardial Damage
and Evaluating the Efficacy of Coronary Artery Bypass Grafting (CABG)
Fig. 1 Serum levels of Hsp 70, IgE, and MMP-9 in the different groups.
the release of inducible Hsp 70 into the circulation in CPK-MB and LDH enzymes levels in the pre-operative patients with minor myocardial damage due to
ischemic patients. Absence of significant changes in coronary insufficiency, even without myocardial
the cardiac enzymes levels between the different infarction. It has been reported that just ischemia could
groups was in agreement with the results obtained by induce Hsp 70 in myocardial cells, Hsp 70 mRNA
Batker et al. [20], who measured CK-MB synthesis increased when coronary flow is gradually
concentration in patients with or without ischemic reduced from normal to zero flow levels. Anaerobic
heart disease of different etiologies. The CK-MB metabolism during ischemia causes depletion of
concentration was the same in patients with stable intracellular ATP which plays an important role in
angina as in those without ischemic heart disease. triggering the HSF1-DNA binding activity [16]. The
In another study of patients with severe stable angina, heat shock transcription factor 1 (HSF1) is a
myoglobin concentration, and the cardiac enzymes, transcriptional activator protein which regulates the
total CK activity, and CK-MB concentration did not inducible synthesis of Hsps [17]. Once activated HSF1
increase in relation to either spontaneous or monomers start to oligomerize as homotrimers, which
exercise-induced ischemia, despite significant ST then bind to an upstream sequence-specific motif, heat
segment depression observed in all patients [21]. shock element, in the promoter of all stress-inducible
Serum levels of IgE, also, showed a significant Hsp genes [18].
elevation in the preoperative samples, when compared It has been hypothesized that anaerobic metabolism
with both post-operative and control ones, it is likely, is a strong stimulus for Hsp 70-gene transcription,
however, that these results are applicable only to since the cessation of anaerobic metabolism is
patients with severe debilitating angina pectoris those associated with complete shutdown of Hsp 70 mRNA
with symptoms severe enough to consider or warrant synthesis, which may explain its return to normal levels
coronary bypass operation. In contrast to our results after performing CABG surgery [19]. Also, Hsp 70
Korkmaz et al. [7] reported that IgE levels were found synthesis could be correlated with the onset of
to be significantly higher only in patients with unstable anaerobic metabolism, but not with enzyme leakage
angina and acute myocardial infarction compared to from the tissue [19], and this explains the elevated Hsp
the patients with stable angina pectoris and controls.
70 levels without an associated increase in the This elevation in total serum IgE levels could be
The Role of Heat Shock Protein 70, IgE and MMP-9 in Detecting Early Minor Myocardial Damage
and Evaluating the Efficacy of Coronary Artery Bypass Grafting (CABG)
explained by the findings of Szczeklik et al. [8], who therefore, so it is supposed that augmented enzymatic reported that IgE may act in humans as an acute phase
activity does not necessarily correspond to increase in protein in response to tissue injury.
its serum level.
The role of IgE-mediated events in the pathogenesis Cardiac surgery, in particular cardiopulmonary of cardiovascular disease may be, in part, mediated
bypass in CABG and cardioplegia, has been reported to through mast cells activation, it has been observed that
trigger myocardial inflammation and apoptosis. This increased numbers of cardiac mast cells were found in
surgery-related inflammatory reaction appears to be of human ischemic hearts [22]. It has been suggested that
extreme complexity with regard to its molecular, in myocardial ischemia circulating IgE sensitizes mast
cellular and tissue mechanisms [28]. cells in the ischemic myocardium; this facilitates
The molecular chaperone Hsp 70 has been detected release of chemical mediators [8]. This paracrine
both in the myocardium and in the circulation after release of cytokines regulates fibroblast phenotype and
CABG. It was reported that serum Hsp 70 reaches its MMP activity [23]. For example, mast cells contain
peak levels by about five hours post-operatively, and substantial stores of tumor necrosis factor-alpha, a
returns to its normal pre-operative levels by about one cytokine capable of inducing MMP synthesis. Also,
day after surgery [29].
human cardiac mast cells contain tryptase, chymase, Systemic blood levels for MMP-9 increase carboxypeptidase, and cathepsin G [24], these enzymes
significantly from baseline values during CABG [30] have been implicated as being involved in the MMP
and remain elevated until 30 minutes after activation cascade by several in vitro studies [25].
cardiopulmonary bypass, MMP-9 levels tend to In the current study, serum levels of MMP-9 showed
decrease toward normal values during the no significant difference between the different groups,
postoperative period, but there is a significant the present findings are consistent with the results of
variability in this response [31].
the study done by Kai et al. [26], who reported that Also, serum IgE that begins to rise shortly after the MMP-9 level in patients with stable angina was not
operations reaches a peak by the fifth day, and then significantly different from that of control, also
gradually declines [7]. In order to avoid any changes in MMP-9 levels measured before, immediately after and
the measured parameters, it could be related to the
1 h after the treadmill exercise test did not change surgical process itself, and not due to changes in the during the exercise test, although exercise induced
myocardial status, the second blood sample was taken angina and typical ischemic ECG changes were
three weeks after surgery.
documented.
5. Conclusion
The possibility that ischemia may cause release and activation of MMPs in the myocardium could not be
On the basis of these results, it could be concluded denied. However, it must be recognized that the release
that, Hsp 70 and IgE might be used as markers for and activity of MMPs involve a highly detection of early minor myocardial damage, and compartmentalized process and that blood MMP levels
coronary insufficiency with less overt damage than likely reflect only spillover from the interstitial
myocardial infarction, as significant changes in their compartment [27]. Furthermore, it is known that all
levels appear before occurrence of in any changes in MMPs require activation from precursors to attain
the levels of MMP-9, CPK-MB and LDH. Besides, enzymatic activity. The antibodies available for serum
Hsp 70 and IgE returning to the normal levels after MMP immunoassays do not distinguish the active form
CABG surgery, suggests that they could be helpful to of these enzymes from their proenzyme forms;
evaluate the effect of CABG surgery.
The Role of Heat Shock Protein 70, IgE and MMP-9 in Detecting Early Minor Myocardial Damage
and Evaluating the Efficacy of Coronary Artery Bypass Grafting (CABG)
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Molecular Cloning of PhoR Sensor Domain from Mycobacterium tuberculosis for Structure-Based Discovery of Novel Anti-Tubercular
1 2 Aldina S. Suwanto 1 , Ihsanawati and Ernawati Arifin Giri-Rachman 1. School of Life Sciences and Technology, Bandung Institute of Technology, Bandung 40132, Indonesia
2. Biochemistry Research Group, Faculty of Mathematics and Natural Sciences, Bandung Institute of Technology, Bandung 40132, Indonesia
Received: March 04, 2011 / Accepted: May 31, 2011 / Published: March 30, 2012.
Abstract: The emergence of multidrug-resistant strains (MDR-TB) and extensively drug-resistant strains (XDR-TB) has fuelled the quest for novel drugs and drug targets for its successful treatment. One of the potential candidates as novel TB drug target is the PhoR sensor domain, an extracellular domain of PhoR histidine kinase. PhoR sensor domain is part of the two-component system PhoR-PhoP that senses environmental stimuli and relays the signal to control the expression of 78 virulent associated genes in Mycobacterium tuberculosis . 3D structure of the PhoR sensor domain will facilitate the structure based drug discovery of novel anti- tubercular. In this study, we successfully predicted and isolated the gene encoding PhoR sensor domain from Mycobacterium tuberculosis H37Rv, cloned it in pGEM-T vector and subcloned it in pRSET emGFP expression vector. PhoR sensor domain was successfully cloned and would be used for further expression, purification and crystallization studies.
Key words: Sensor domain PhoR, tuberculosis, two-component system.
1. Introduction In response to a particular environmental stimulus HK will transfer a phosphate to a histidine residue on RR.
Sensing and responding to extracellular conditions The RR is commonly a transcription modulator and its are very essential for the survival of bacteria. To activation by phosphorylation leads to the expression respond appropriately to different environmental of genes required to adapt the environment (Fig. 1a). changes, bacteria have evolved two-component signal Two-component systems are responsible for transduction systems (TCS). A typical TCS is governing responses to many stresses relevant to composed of a sensor histidine kinase (HK) and a pathogenesis such as chemical assaults, nutrient response regulator (RR) that interacts with the deprivation, osmotic stress and macrophage survival. phosphorylated HK. Phosphorylated RRs bind to the In some cases they are responsible for sensing upstream-regulatory region of pathogenic genes and conditions that cause microbes express virulence control their expression [1, 2]. factors and adopt a pathogenic lifestyle [3, 4]. Histidine kinases usually function as dimeric So far, 11 TCS have been identified in proteins. The signal transduction cascade starts with Mycobacterium tuberculosis (MTB), the causative the sensing of a signal ligand in the HK sensor domain. agent of tuberculosis. Among those 11 TCS, the
two-component system PhoR-PhoP is the one whose Corresponding author: Ernawati Arifin Giri-Rachman,
disruption was shown to affect MTB ability to replicate Ph.D., research field: genetics and molecular biotechnology.
E-mail: [email protected]. in cellular and animal models most dramatically.
Molecular Cloning of PhoR Sensor Domain from Mycobacterium
Tuberculosis for Structure-Based Discovery of Novel Anti-Tubercular
Fig. 1 Signal transduction mechanism of two-component system PhoR-PhoP and inhibition of PhoR sensor domain.
a: normal signal transduction. Binding of signal ligand to the dimeric sensor domain triggers autophosphorylation of PhoR. Subsequently, phosphoryl group is transferred from PhoR to response regulator PhoP. Phosphorylated PhoP activates DNA transcription. b: Blockage of first step in signal transduction system through inhibition of PhoR sensor domain of inhibition. In this case, sensor domain does not sense the signal and it would turn off expression of genes under regulation of PhoP regulon, modified from Matsushita and Janda [3].
PhoR-PhoP TCS controls the expression of 78 pathway and subsequently stop the expression of virulent-associated genes of MTB (approximately 2%
virulent genes (Fig. 1b). Moreover, the absence of of the coding capacity of MTB genome). Genes
sensor histidine kinases in humans can be exploited regulated by PhoP in MTB include those required for
for the development of a safer drug candidate with hypoxia adaptation, aerobic/anaerobic respiration, reduced side-effect [9].
genes within region of difference 1 (RD1), genes The three dimensional (3D) structure of the PhoR encoding stress protein and genes involved in lipid
sensor domain will facilitate the structure based drug metabolism. A number of investigations show that
discovery of novel anti-tubercular drugs targeting inactivation of PhoR-PhoP system leads to significant
specifically on PhoR sensor domain. The field of growth attenuation. Also, biochemical results reveal
structure based drug discovery itself is a rapidly that PhoP regulon regulates biosynthesis of sulfolipids
growing area in which many successes have occured (SL), diacyltrehaloses (DAT) and polyacyltrehaloses
in recent years. Structural information has the (PAT) and absence of these complex lipid molecules
potential to improve the quality of new drugs as well would be a major reason for MTB attenuated growth
as save time and money compared to traditional in a mouse model [5-8].
trial-and-error methods [10, 11]. PhoR sensor domain is the extracellular domain of
In this paper, we predicted residues of PhoR protein histidine kinase PhoR that senses environmental
that corresponds to the extracellular sensory domain, stimuli and relays the signal to control the expression
isolated the gene encoding PhoR sensor domain, of genes under PhoP regulon. Thus, PhoR sensor
cloned it into pGEM-T vector and subcloned into domain has emerged as an attractive drug target since
pRSET emGFP expression vector. The molecular it will block the first step in the signal transduction
cloning work done in this research is the first step of
Molecular Cloning of PhoR Sensor Domain from Mycobacterium Tuberculosis for Structure-Based Discovery of Novel Anti-Tubercular
the roadmap to determine the crystal structure of PhoR
2.3 Cloning into pGEM-T Vector and Subcloning into sensor domain and subsequently to design novel
pRSET emGFP Expression Vector anti-tubercular drugs targeting on PhoR sensor domain.
The amplified DNA was then ligated into the
2. Material and Methods
ampicillin-selectable pGEM-T vector (Promega, USA). The plasmid was introduced into CaCl 2
2.1 In Silico Characterization prepared E. coli TOP10 competent cells by heat shock
The sequence of PhoR from Mycobacterium method. Bacterial colonies were screened by the tuberculosis (MTB) strain H37Rv was retrieved from
blue-white screening method using isopropyl- β- GenBank (accesion number NC_000962.2). HK class
thiogalactopyranoside (IPTG) and X-gal. Several was determined using conserved domain database
colonies were selected and cultured for 12 hours. (CDD) BLAST and compared to E. coli EnvZ (HK
Plasmid molecules were harvested by alkaline lysis class I model) dan CheA (HK class II model)
method [15] and cleaved with restriction enzymes according to Dutta et al. [12].
Eco RI and NcoI. pRSET emGFP expression vector PhoR sensor domain topology was predicted with
(Invitrogen, USA) was linearized using the same DAS Transmembrane Prediction and TopPred [13].
enzymes. Subsequently, the PhoR sensor domain Based on the predicted transmembrane segments and
fragment was ligated into corresponding sites of the HK class, residues corresponding to PhoR sensor
linearized pRSET emGFP to obtain pRSETSensPhoR. domain were determined. Geneious motif analysis [14]
The resulting final construct (Fig. 2) pRSETSensPhoR was carried out to characterize the protein motif.
was transformed again into CaCl 2 prepared E. coli TOP10 competent cells. Cells containing the plasmid
2.2 Gene Isolation from Mycobacterium tuberculosis were grown in LB medium containing ampicillin at H37Rv Genome
37 °C. Plasmid was harvested by alkaline lysis method Using appropriately designed 5’ and 3’ primers, we
and cleavage with restriction enzymes EcoRI and
amplified segment of Mycobacterium tuberculosis
I was carried out to screen plasmids containing the strain H37Rv genome that corresponds to residues
Nco
360 bp fragment of PhoR sensor domain. Sequencing 36-155 of PhoR, indentified here as PhoR sensor
was performed to confirm the final construct. domain by previous in silico analysis. The forward
3. Results and Discussions
primer 5’-GAA TTC GTC ACC TCG ATG TTG CAG CA contains a unique EcoRI site added
3.1 Characteristics of PhoR Sensor Domain (underlined). The reverse primer 5’-CCA TGG TTA
On the basis of domain organization, histidine GAC GTC GGC CAG ATC AAT G contains a unique
kinases (HKs) have been grouped into two major Nco
I site (underlined) and UAA stop codon (bold) classes. In class I, which are exemplified by EnvZ the added. Amplification was performed in a total volume
H box-containing region is directly linked to the of 25 µL containing the following PCR mixture.
region that contains the other four conserved boxes in Template DNA, 12.5 pmol of each primer, 0.2 mM
HK (H, N, G1, F and G2 boxes). However, in class II, dNTPs, 1× PCR Buffer, 2.5 unit of Taq DNA
like CheA, the H box-containing region is distant from
the catalytic domain by having distinct domain as follows : 94 °C, 3 min (initial denaturation step);
polymerase, and 1.5 mM MgCl 2 . PCR conditions were
insertions between them (Fig. 3) [12].
94 °C, 1 min, 64 °C, 1 min, 72 °C, 2 min for 25 cycles; CDD BLAST results of PhoR and representatives
72 °C, 7 min using a thermocycler. Amplified DNA of HK class I and II (EnvZ and CheA correspondingly) was purified and confirmed by sequencing.
are shown in Fig. 4.
Molecular Cloning of PhoR Sensor Domain from Mycobacterium
Tuberculosis for Structure-Based Discovery of Novel Anti-Tubercular
Fig. 2 Schematic construction of vectors used in molecular cloning of PhoR sensor domain from Mycobacterium tuberculosis. The 360 bp sensor domain PhoR PCR product was first ligated into pGEM-T vector (upper figure) using restriction enzyme site EcoRI and NcoI. Sensor domain PhoR was subsequently subcloned into expression vector pRSET emGFP (lower figure).
Fig. 3 Histidine kinase class I and II. Schematic diagram of domain organization and structure of representatives of HK class I and II are presented. HK class I is represented by E. coli EnvZ and class II is represented by E. coli CheA [12].
Fig. 4 Conserved domain database BLAST of Mycobacterium tuberculosis PhoR, E. coli EnvZ (class I HK), and E. coli CheA (class
II HK). Results showed that PhoR has similar motifs and structural organization to EnvZ and thus is member of class I HK.
Molecular Cloning of PhoR Sensor Domain from Mycobacterium Tuberculosis for Structure-Based Discovery of Novel Anti-Tubercular
CDD BLAST clearly showed that MTB PhoR has predominantly hydrophobic residues separated by the same structural domain organization with E. coli
polar connecting loops as transmembrane helices EnvZ (HK class I) with the dimer interface and
[13]. Hydrophobicity profile (Fig. 5) showed three histidine phosphorylation site located next to the ATP
segments with predominant hydrophobicity binding site (catalytic domain). E. coli CheA as a
corresponding to the three transmembrane helices representative of HK class II has a different domain
predicted. Result of this transmembrane prediction organization.
and topology prediction (Fig. 6) matches with the Class I HKs are usually homodimeric membrane
HK class I domain organization as described by proteins. Each monomer contains a short N-terminal
Sevvana et al. [16].
cytoplasmic domain followed by a transmembrane α The N-terminal is located in cytoplasm, followed
helix and an extracellular sensory domain that is by the first transmembrane helix, sensor domain, connected via a second transmembrane α helix to the
second transmembrane helix, and the cytoplasmic cytoplasmic C-terminal HK domain [16]. Thus, in HK
domain. Thus, MTB PhoR sensor domain lies from class I, the extracellular sensory domain is the
residue 36-155, which is between the two first segment between two transmembrane helices.
transmembrane helices.
Transmembrane prediction indicates the presence Geneious motif analysis result (Fig. 7) showed that of three transmembrane segments in PhoR protein
MTB PhoR have a mixture of α helix and β sheets. from MTB strain H37Rv. The first segment Recent structural study have revealed three classes of corresponds to residue 15-35 of PhoR, the second
HK sensor domain crystals: mixed alpha-beta folds segment corresponds to residue 156-176 of PhoR and
(PDC domain), all alpha folds, and sensor domains the third segment is residue 436-457. Surface
that show a similar fold to periplasmic binding transmembrane prediction predicts streches of 15-30
proteins [17].
Helix Begin-End Score Certain 1 15-35 1.979 Certain 2 156-176 1.590 Certain 3 436-457 1.006 Certain
Fig. 5 Hydrophobicity profile of PhoR sensor domain and predicted transmembrane helices using DAS and TopPred.
Molecular Cloning of PhoR Sensor Domain from Mycobacterium
Tuberculosis for Structure-Based Discovery of Novel Anti-Tubercular
Fig. 6 Topology prediction of PhoR protein from MTB. In Class I HK, sensor domain lies between two transmembrane helices (shown by arrow).
Fig. 7 Geneious motif analysis result of PhoR sensor domain.
Based on geneious motif prediction, we can assume that PhoR sensor domain would be most likely to have
a PDC domain. PDC domain is a mixed alpha-beta structural class of sensor domains, in reference to the initial three extracellular sensor domains PhoQ, DcuS and CitA for whose structures were first determined. Ribbon diagrams of DcuS and DctB having PDC domain were shown in Fig. 8 [17].
3.2 Molecular Cloning
Based on in silico analysis, suitable primers were
Fig. 8 Ribbon structure of DcuS and DctB with the typical
generated for isolation and amplification of PhoR PDC domain. PDC domain have been observed in all crystals
of mixed-alpha-beta HK sensor domain [17].
sensor domain of MTB. We successfully isolated the gene encoding PhoR sensor domain from MTB strain
facilitate cloning into expression vector pRSET emGFP H37Rv by means of PCR. The PCR product is about
UAA stop codon was also added to the reverse primer. 360 bp long and related to residue 36-155 of PhoR.
Analysis of agarose gel electrophoresis showed that a Restriction sites for cleavage with restriction enzymes
single band between 300 and 400 bp was obtained Eco RI and NcoI were added to the designed primer to
(Fig. 9a). This matches the length of the desired
Molecular Cloning of PhoR Sensor Domain from Mycobacterium Tuberculosis for Structure-Based Discovery of Novel Anti-Tubercular
Fig. 9 a: Agarose gel electrophoresis analysis of PCR product. Lane 1 showed a single band with length around 300-400 bp. The desired PhoR sensor domain is about 360 bp long. Lane 2 is negative control. M: DNA marker, 100 bp. b: Restriction analysis of recombinant plasmid pGEM-T sensor domain PhoR. Lane 1 showed circular pGEM-T sensor domain PhoR. Lane 2 showed restiction fragment of pGEM-T sensor domain PhoR cut with EcoRI and NcoI. A fragment between 250 and 500 bp was obtained. pGEM-T backbone is ~3,000 bp. M: DNA marker, 1 kb. c: Restriction analysis of final construct pRSET SensPhoR. Lane 1 showed circular pRSET SensPhoR plasmid. Lane 2 showed restiction fragment of pRSET SensPhoR cut with EcoRI and NcoI. A fragment between 250 and 500 bp was obtained. pRSET emGFP backbone is ~3,600 bp. M: DNA marker, 1 kb.
Fig. 10 Alignment of the final construct (pRSETSensPhoR) sequencing result and PhoR sensor domain from MTB using ClustalW2 [18]. Sequencing result showed pRSET emGFP expression vector with start codon, 6× His Tag, T7 gene 10 leader, sensor domain PhoR between restriction site of EcoRI and NcoI, stop codon and emGFP sequence. This sequencing result confirmed succesful cloning. Sensor domain PhoR in the final construct matches 100% with the sequence of PhoR sensor domain from MTB H37Rv.
PhoR sensor domain which is around 360 bp. Eco RI and NcoI (Figs. 9b and 9c) was carried out Sequencing result confirmed that the fragment is PhoR
following succesful transformation of recombinant sensor domain of MTB.
plasmid into E. coli host cell.
PCR product was also succesfully cloned into Sequencing of the final recombinant plasmid pGEM-T vector and subcloned into pRSET emGFP
pRSETSensPhoR confirmed succesful cloning (Fig. 10). vector. Restriction analysis with restriction enzyme
The presence of start codon, 6× His Tag and T7 gene
Molecular Cloning of PhoR Sensor Domain from Mycobacterium
Tuberculosis for Structure-Based Discovery of Novel Anti-Tubercular
10 leader in the expression vector was also confirmed. structure of virulence-associated response regulator PhoP of Mycobacterium tuberculosis: Role of the linker region
Reading frame is corrected and stop codon was also on regulator-promoter interaction, Journal of Biological
succesfully added before the emGFP sequence. Chemistry 285 (45) (2010) 34309-34318. The final construct obtained in this study will be
[6] J.G. Asensio, C. Maia, N.L. Ferrer, N. Barilone, F. Laval, further expressed in E. coli expression system, C.Y. Soto, N. Winter, M. Daffe, B. Gicquel, C. Martin, M. Jackson, The virulence-associated two-component purified and crystallized to obtain the three
PhoP-PhoR system controls the biosynthesis of dimensional structure of PhoR sensor domain.
polyketide-derived lipids in Mycobacterium tuberculosis, Journal of Biological Chemistry 281 (3) (2006) 1313-1316.
[7] J.G. Asensio, S. Mostowy, J. Harders-Westerveen, K. Huygen, R. Hernandez-Pando, J. Thole, et al., PhoP: A The gene encoding PhoR sensor domain was
4. Conclusion
missing piece in the intricate puzzle of Mycobacterium succesfully isolated from Mycobacterium tuberculosis
tuberculosis virulence, PloS ONE 3 (10) (2008) e3496. [8] S.B. Walters, E. Dubnau, I. Kolesnikova, F. Laval, M. H37Rv by means of PCR, further cloned into
Daffe, I. Smith, The Mycobacterium tuberculosis PhoPR pGEM-T vector and subcloned into expression vector
two-component system regulates genes essential for pRSET emGFP. The insert consisting of 360 bp DNA
virulence and complex lipid biosynthesis, Molecular Microbiology 60 (2) (2006) 312-330.
encoding 120 amino acids was confirmed by [9] J.S. Tyagi, D. Sharma, Signal transduction systems of sequencing. PhoR sensor domain was successfully
mycobacteria with special reference to M.tuberculosis, cloned and would be used for further expression,
Current Science 86(1) (2004) 93-102. [10] K.E. Goodwill, M.G. Tennant, R.C. Stevens, High-throughput
purification and crystallization studies as a target for X-ray crystallography for structure-based drug design, Drug
structure-based discovery of novel anti-tubercular Discovery Today 6 (15) (2010) S113-S118. drugs.
A.C. Anderson, The process of structure-based drug design, Chemistry and Biology 10 (2003) 787-797.
Acknowledgment
[12] R. Dutta, L. Qin, M. Inouye, Histidine kinases: Diversity of domain organizations, Molecular Microbiology 34 (4) The authors would like to extend gratitude to
(1999) 633-640.
Ms. Vitri Dwi Astuti for providing Mycobacterium [13] M. Cserzo, E. Wallin, I. Simon, G. Von Heijne, A. tuberculosis H37Rv genome for this research.
Elofsson, Prediction of transmembrane alpha-helices in procariotic membrane proteins: The dense alignment
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Journal of Life Sciences 6 (2012) 276-282
Effect of Cement Dust Pollution on the Yield and Quality of Ficuscarica L. Fruits
Amal Mohamed Abdel-Rahman Department of Botany, Faculty of Science, University of Alexandria, Alexandria, P.O.B. 21511, Egypt
Received: August 09, 2011 / Accepted: October 13, 2011 / Published: March 30, 2012.
Abstract: The present study was carried out on Ficuscarica L. cultivated in the northwestern desert of Egypt. Plant materials (leaves and fruits) were collected from three polluted locations at a distance of 500-700, 1,000-1,250 and 3,000-3,500 m respectively away from the cement factory at El-Hammam city, and a location of relatively clean air considered a control at 5,000-6,000 m away from this
factory. The deposit cement dust washed from the surface leaf area of plant study was found to be 4.96, 4.21, 0.51 and 0.29 µg/cm 2 at the four locations, respectively. Cement in more polluted locations increased mortality of young branches leading to a reduction in the height and yield of fig trees. The deposition of cement pollutants tothe loamy sandy soil of the present study alteredsoil chemical characteristics. The results showed that, biomass of fruits/tree, number of branches/tree and number of fruits/branch in polluted locations were significantly lower than those of the control one. Cement dust decreased leaf total chlorophyll content leading to a reduction in the economic yield (up to 50%). Metabolic constituents (carbohydrates, proteins, amino acid and proline) and essential elements (Fe, Mg, Na, Ca, and K) were studied in two types of fruits on fig trees (mature and premature). Thallium as a toxic metal was predicted in edible mature fruits, and the results showed that the concentration of thallium parts per billion (ppb) in polluted locations was significantly higher than those of the control one. The results revealed that fruits of fig plants at polluted sites showed quantitative and qualitative deteriorations.
Key words: Ficuscarica L., cement dust, metabolic constituents, thallium, yield.
1. Introduction effects on soil quality and fertility [3-7], whereas soil surrounding cement factories, especially downwind
Air pollutants, responsible for vegetation injury and areas, exhibit elevated pH level [8-10]. In a soil crop yield losses are causing increased concern [1]. composition, it was found that there were elevated Cement dust is one of the most important pollutants in levels of chromium, silica, iron, calcium and thallium the environment that poses a threat to the proper with contamination level decreasing dramatically with functioning of plant near the cement factories [2]. distance from the factories. Thallium in the soils is Industries emit toxic substances that adversely affect derived from burning of coal at cement works in man’s food supply by affecting growing plants that are various countries. It is probable that the contamination particularly susceptible to pollution. The particulate is derived from the raw materials used to make cement, dust falling on the leaves of plants have been found to rather than from the burning of coal in itself [11]. affect photosynthesis, stomatal functioning, These compositions affect vegetative growth [12, 13]. productivity, pigment and metabolic constituentsand Ficuscarica L., an economically important crop, has also cause foliar injuries, reduction in yield and uptake been chosen to study the effect of cement dust pollution and accumulation of elements from soilbeside its major on fig leaves, plant productivity (yield) and nutritional
quality of edible part (syconium), since only few Corresponding author: Amal Mohamed Abdel-Rahman,
studies have been conducted of particulate pollution on Ph.D., lecturer, research field: ecophysiology. E-mail:
economic fruit crop plants.
Effect of Cement Dust Pollution on the Yield and Quality of Ficuscarica L. Fruits
2. Materials and Methods
Phytomass of fig fruits were determined. Samples of leaves were collected from these trees and washed
The study area was located between burg El-Arab gently by distilled water which was collected in a clean, and Hammam cities in the northwestern desert of Egypt weighed beaker and then evaporated to determine the and about 60 km west of Alexandria. weight of washed dust as µg/cm 2 of the leaf area which Four orchards of Ficuscarica L. were selected at was measured using a digital planimeter (Bolab model four different locations downwind from a cement
40) with a sensitivity of 0.1 cm 2 . The washed leaves factory in the study area. The first location was about
were collected for determination of pigment contents 500-750 m south from the factory, the second location
according to Metzner et al. [22].
was about 1,000-1,250 m east from the factory, the Soil samples were collected from several random third location was about 3,000-3,500 m from the locations in each orchard for determination of physical factory, and the fourth location (control) was about
and chemical characters.
5,000-6,000 m far from the factory where the cement Treatment of data: analysis of variance was applied dust load was nearly zero. In each location selected one way (F-test) to assess the significance of variations randomly ten trees as accounting the number of branches in different variables in different locationsand t-test in each tree and the number of fruits in each branch. applied to assess the significance of variation between Two types of fruit samples (mature, premature) were
mature and premature fruits for each variable.
collected at maturity stage in summer from random trees approximately at the same age for measurement
3. Results and Discussion
and analysis. All samples were prepared and total available
The present study showed that pH of soil solution in carbohydrates (TAC) and total soluble sugars (TSS)
polluted locations 1, 2 and 3 were slightly more were analyzed according to the methods described by
alkaline (8.0, 8.1, 7.8) compared with the control (7.7) Naguib et al. [14, 15]; total protein (TP) and soluble
and the electrical conductivity (EC) also increased by protein (SP) were analyzed according to Lowry et al.
cement dust (36.0, 32.8, and 4.9 ds/m) for locations 1, 2 [16, 17]; total free amino acids (AA) according to Ya et
and 3 respectively and for the control (1.9 ds/m).
al. [18]; proline according to Bates et al. [19]; some 2- The levels of Na, K, Ca, Mg, Cl and SO
4 were nutrient elements such as Fe, Mg, Na, Ca, K were
higher in the polluted soil than in the control soil, while analyzed according to Allen et al. [20] and thallium - there was no difference between the levels of HCO and
according to Barnes Karen [21].
CO 3 2- in polluted and control soils (Table 1).
Table 1 Chemicalanalysis of the soil samples at different locationsin the study area.
Locations
1 2 3 Cont. pH 8.0 8.1 7.8 7.7 EC (ds/m)
K + 5.6 5.1 0.5 0.8 Cation (meq/L)
Ca ++ 130
85 11.8 5.0 Mg ++ 70 75 9.0 3.6
Cl - 338 294 37.5 12.5 HCO - 3 6.0 6.0 8.0 6.0
Anion (meq/L)
CO 2- 3 -
tr.
tr.
SO 4 2- 26 35 3.5 0.5
Effect of Cement Dust Pollution on the Yield and Quality of Ficuscarica L. Fruits
These results agree with the results obtained by that once inside the cells, aluminum could inhibit root Sivakumar et al. [23, 24], who reported that
absorption and growth in both aluminum sensitive and atmospheric pollution produced by cement works
tolerant plants [28]. Cement dust may change leaf
physiology leading to reduced productivity [3]. silicon and Na 2 SO 4 . It also containstraces of heavy
include high level of CaCO 3 and oxide of potassium,
Photosynthetic pigments (chl a, chl b, carotenoids) metals and halogens like thallium and iodine, this dust
were declined with cement dust accumulation (Fig. 1) falling on the soil caused a shift in pH to the alkaline
causing an increase in chl a/b ratio in location 1 (3.5) side and increase in soluble salts in it.
while decrease in the others locations. chl (a+b) also The data listed in Table 2 showed that the amount of
decreased with increased cement dust about 52% and deposited dust varied significantly (P < 0.05) 27% in locations 1and 2 respectively and about 8% in according to the distance of the four locations (1, 2, 3
location 3 (Fig. 2).
and control) from the cement factory (4.96, 4.21, 0.5, This leads to a decrease in photosynthetic rate and
0.29 µg/cm 2 ) of leaf area respectively. quantum yield and could be explained on the bases of Field observations showed that the height of fig trees
quantitative as well as qualitative changes in the were reduced in more polluted locations (1, 2) and
incident light available for photosynthesis in cement fruits of the trees in location 1 not complete their
dusted leaves [29]. These findings confirmed the growth and are released (about 90%) and in location 2
previous results by Singh and Rao [2] on wheat plants
[30] and on Vignamungo, and Nanos and Ilias [3] on location 3 to location 1, number of branches/tree and
(about 40%). Leaf area (cm 2 ) decreased by dust from
olive leaves physiology.
number of fruits/tree exhibited a significant decrease Our results indicate the percentage of chl a/b ratio (P < 0.001) in response to cement dust.
increased in more pollutant location 1 caused by great Fruit phytomass (economic yield) kg.d.wt/tree or
decrease in chl b about 60% compared with its value at kg.f.wt/tree exhibited also a highly significant decrease
control, while a marked decrease in chl a/b ratio was (P < 0.001) in response to increased cement dust. Yield
observed in the other locations 2 and 3. at more polluted locations (1, 2) decreased to about
The changes in chlorophylls a and b are possibly due 0.5%-6.0% respectively and in less polluted location 3
to shading and/or photosystem damage [3]. to about 50% compared to the control. This is in
The average concentration of metabolic constituents agreement with those authors [25-27]. The reduction in
is represented in Table 3.
growth may be due to toxic effect of aluminum which These metabolites varied significantly (P < 0.001) was found in cement dust,and some researchers stated
with the distance from the cement factory according to
Table 2 Variations in Different Parameters of Ficuscarica L. at different Locations in the Study Area.
Locations
F (P) 1 2 3 Cont.
No. of ranches/tree ± SE
51.30 c ± 7.76 60.80 d ± 4.64 162.248* (< 0.001) No. of fruits/branch ± SE
5.30 a ± 1.89
21.70 b ± 8.84
34.50 d ± 3.50 162.182* (< 0.001) Leaf area (cm 2 ) ± SE
3.20 a ± 1.62 13.20 b ± 4.61
21.60 c ± 2.67
177.71 a ± 8.93 274.93 b ±23.24 292.73 b ±13.61 282.63 b ±25.98 6.569* (0.001) Dust wt (µg/cm 2 ) of leaf area
4.96 4.21 0.51 0.29 - Yield/tree on fresh wt basis (kg. f.wt)
0.60 5.50 50.00 92.50 - Yield/tree on dry wt basis (kg.d.wt) 0.09
0.95 8.87 18.82 - F : F-test (ANOVA); *: Statistically significant at P ≤ 0.05; Different symbol are statistically significant.
Effect of Cement Dust Pollution on the Yield and Quality of Ficuscarica L. Fruits
chlorophyll a
(m 0.6
chlorophyll b
Fig. 1 Variations in the Content of Pigments at different locations in the Study Area.
Fig. 2 Variations in the chl.a/b ratio and chl a+b at different locations in the Study Area.
Table 3 Variations in the Mean Concentration ± SE of different Metabolites in Fruits of Ficuscarica L. at different Locations in the Study Area.
1,580.538* (< 0.001) TAC Mature 384.54 (mg/g.d.wt) a ± 2.87
Premat. 174.26 a ± 1.05
903.747* (< 0.001) t (P)
Premat. 46.77 ab ± 0.26
1,748.499* (< 0.001) t (P)
Mature 316.25 a ± 0.86
(mg/g.d.wt)
16.271* (0.010) TP
Premat. 111.57 a ± 0.71
(mg/g.d.wt)
400.374* (< 0.001) t (P)
Mature 192.45 a ± 1.11
Premat. 81.59 a ± 1.16
Mature 157.12 (mg/g.d.wt) a ± 1.00
226.085* (< 0.001) t (P)
4,445.195* (< 0.001) Amino acids
Premat. 4.30 a ± 0.04
21.55 (mg/g.f.wt) d ± 0.06 1,654.644* (< 0.001) t (P) 3.518* (0.002)
Mature 8.165 a ± 0.07
Premat. 0.61 a ± 0.01
6,406.100* (< 0.001) (mg/g.f.wt) t (P) 2.968* (0.006)
Mature 1.30 a ± 0.01
F : F-test (ANOVA); t: Student t-test; *: Statistically significant at P ≤ 0.05; Different symbol are statistically significant.
Effect of Cement Dust Pollution on the Yield and Quality of Ficuscarica L. Fruits
F -test and also between two types of fruits cement dust affected on nutrient elements (mature-premature) according to t-test.
accumulation in edible mature fruits (Table 4). TAC and TSSexhibited a decrease trend in response
Nutrient elements, such as Fe, Mg and Ca were more to increase in cement dust from location 3 to location 1.
accumulated in mature fruits in response to cement dust For mature fruits a gradual decrease about 25% in
stress, which agrees with Ade-Ademilua [25] who location 1 (the most polluted one) but for premature
reported that cement pollution increased the fruits the maximum decrease in the concentrations of
concentration of Fe and Mg in leaves and stems of TAC and TSS occur in location 3 (the less polluted one)
Celosia argentea , and with Jurga [6] who reported that about 43% and 73% respectively.
apple tree growing in the vicinity of cement factory TP and SP for mature fruits increased by the cement
sustains greatly accumulate Ca and Mg, but notably dust pollution except in location 2 and for premature
nutrient elements such as Na and K which were less fruits TP increased with cement dust pollution except
accumulated in mature fruits in response to cement dust in location 1, while SP decreased by increase in cement
stress. Seufert [32] reported that higher concentrations dust pollution. Amino acids and proline are also
of air pollutants may increase or decrease the uptake of decreased more than 50% with increase in cement dust
especially in locations 1 and 2, whereas dust input in
these locations was the maximum (4.96, 4.21 µg/cm 2 of
leaf area). These results agree with those of many investigations on different plant species who reported that, the metabolites decreased in dusted plants as compared to control [2, 30].
Essential elements, such as Fe, Mg, Na, Ca, and K play an important role in plant nutrition and can affect
crop yields when not present in appropriate Fig. 3 Variations in the mean concentration (ppb) of thallium
concentration levels [31]. The present study shows that,
in mature fruits at different locations in the study area.
Table 4 Variations in the mean concentration ± SE of some elements in fruits of Ficuscarica at different locations in the study area.
Premat. 0.07 a ± 0.03
0.03 (mg/g.d.wt) b ± 0.00
36.194* (< 0.001) t (P) 1.944 (0.070)
Fe Mature 0.06 a ±0.00
0.03 b ± 0.00
0.02 c ± 0.00
87,244.914* (< 0.001) Mg Mature 1.06 (mg/g.d.wt) a ± 0.00
Premat. 2.71 a ± 0.01
16,909.523* (< 0.001) t (P) 3.702* (0.003)
Premat. 9.12 a ± 0.00
253,663* (< 0.001) (mg/g.d.wt) t (P) 1.679 (0.107)
Mature 5.69 a ± 0.00
Premat. 20.53 a ± 0.05
(mg/g.d.wt) Mature 10.46 ± 0.03
4,979.208* (< 0.001) t (P) 15.627* (< 0.001)
Premat. 5.98 a ± 0.00
(mg/g.d.wt) Mature 8.81 ± 0.01
878,149.61* (< 0.001) t (P) 2.139 (0.053) -test (ANOVA); t: Student t-test; *: Statistically significant at P F: f ≤ 0.05; Different symbol are statistically significant.
Effect of Cement Dust Pollution on the Yield and Quality of Ficuscarica L. Fruits
nutrients by the roots. In addition to polluted soil
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Journal of Life Sciences 6 (2012) 283-286
The Effect of Drought Occurring at Different Growth Stages on Productivity of Grain Amaranth Amaranthus cruentus G6
Silva Grobelnik Mlakar, Martina Bavec, Manfred Jakop and Franc Bavec Faculty of Agriculture, University of Maribor, Pivola 10, Ho če 2311, Slovenia
Received: October 11, 2011 / Accepted: November 16, 2011 / Published: March 30, 2012.
Abstract:
A greenhouse pot experiment was conducted to study the effect of drought induced at different phenological stages on growth, biomass production and yield performance of grain amaranth Amaranthus cruentus G6. After emergence seedlings were exposed to different soil water regimes: constant adequate moisture (W1) and drought (W2) throughout the growing period, drought initiated at crop inflorescence formation (W3), drought condition during pre-inflorescence formation (W4) and treatment W5 where drought condition occurred in the period from the beginning of inflorescence formation to the beginning of flowering. Crop samples were taken at the maturity. The growth and yield performance of amaranth were assessed by measuring root length, stem height and inflorescence length, and by evaluating fresh and dry weight of plant parts, grain yield and harvest index. Drought stress initiated at different phenological stages affected the evaluated morphological parameters, assimilate allocation and grain yield. Drought throughout the growing period resulted in grain and biomass yield reduction for 51% and 50%, respectively. Water deficit during inflorescence formation appears to be critical growing stage influencing grain yield, while soil drying in the vegetative growth stages improve the assimilate allocation to the above-ground biomass and particularly to the grain.
Key words: Grain amaranth, Amaranthus cruentus, drought, biomass allocation, grain yield.
1. Introduction amaranth [4-6] and yield formation of grain amaranth under drought conditions is still unclear.
Soil drought is one of the major limiting factors in Therefore, the objective of the study, which was a crop production and it will become increasingly part of a broader national research project on grain important due to global climate changes. Instead of amaranth, was to quantify the effects of soil water deficit nutritional quality [1], grain amaranth (Amaranthus occurred at different phenological stages on biomass
spp.), as a plant possessing C 4 -photosynthesis pathway,
allocation and productivity of Amaranthus cruentus G6. was also recognized as drought tolerant crop [1].
Johnson and Henderson [3] reported, for examined
2. Materials and Methods
grain amaranth cultivars, mean total water use (TWU)
A pot experiment was conducted in the conditions of of 267 mm and grain water use efficiency (WUE) value plastic greenhouse at the University Agricultural of 5.9 kg·ha -1 ·mm -1 . The values obtained in the study Centre of Faculty of Agriculture, Maribor in 2007. The were comparable to other drought adapted crops. The pipes tubes (75 cm in high, 14 cm in diameter), filed development responsiveness to soil water has only with native top soil were exploited as the experimental been reported in pot experiments on vegetable pots. After pot’s filling grain amaranth Amaranthus
cruentus G6 was over sown and tensimeter tubes (30 Corresponding author: Franc Bavec, Ph.D., professor,
research fields: organic farming, field crop production, cm long) were placed randomly into four pots per alternative crops. E-mail: [email protected].
The Effect of Drought Occurring at Different Growth Stages on Productivity
of Grain Amaranth Amaranthus cruentus G6
treatment. Used genotype was proved as perspective extracting the plant out of tube and washing off the soil. and most suitable for NE Slovenian growth condition
All plant parts were oven-dried at 70 °C for three days, among the tested germplasm [7-9]. According to
and dry weights were determined. Harvest indexes (HI r performance in extremely drought condition of the year
and HI), as the ratios of grain to biomass yield in dry 2003 (crop sown in may yielded 2,032 kg·ha -1 of grain
matter with and without root mass were determined, and 20,457 kg·ha -1 of above-ground biomass), respectively. genotype is also presumed to be drought tolerant
The experiment was arranged in randomized block (Grobelnik Mlakar, unpublished data). Amaranth
design with three replications. Obtained data were seedlings were thinned to one plant per pot in the stage
subjected to analysis of variance (ANOVA) using of emergence. After sowing pots were subjected to two
Statgraphics Centurion XV (Statpoint Technologies, different soil water tensions (SWT): -0.4 MPa (SWT 1)
Herndon, USA) to ascertain significant differences considered as adequate moisture, and -0.7 MPa (SWT
between treatments (P ≤ 0.05). Differences between
2) considered as drought condition. Soil water regime means were revealed by Duncan’s multiple range test (SWR) treatments, comprised of a given tensions,
initiated at different phenological stages were: constant
3. Results and Discussion
adequate moisture (W1) and drought (W2) throughout the growing period, drought initiated at crop
Soil water stress affected all studied parameters inflorescence formation (W3), drought condition until
(Table 2). Plants subjected to soil water stress the inflorescence formation (W4) and treatment W5
exhibitedslight increase in rooting depth. In where drought conditions occurred in the period from
comparison to control (W1), the differences were not the beginning of inflorescence formation to the
significant except for treatment W5, where drought beginning of flowering (Table 1). The stated occurred during inflorescence formation and tap root phenological stages were considered to begin when
was longer for 34%. Results are in accordance to inflorescence was visible and when pollen started to
Johnson and Henderson [3], who reported grain shed on 75% of the plants. Soil water tension was
amaranth apparent ability to respond to water stress monitored every second day during the course of the
also by increasing rooting depth that makes it a experiment using the puncture tensimeter instrument potentially useful crop, where soil moisture conditions
(SDEC France, SMS 2500S) and tap water was added vary considerably among growing seasons. Plant according to soil water retention curve defined by six
growth decreased according to drought duration. points from 2.5 to 4.2 pF.
However, in comparison to constant adequate moisture Nine plants per treatment were taken at the time of
lasted throughout the growing period, the stem height harvest, and root depth, plant height and fresh biomass
was not affected and inflorescence was even longer (separately for root, stem, leaves, inflorescence and
when drought occurred in the period of vegetative grain) were recorded. Root material was obtained by
growth stages (W4). As morphological traits, biomass
Table 1 SWR (W1-W5) were Comprised of Adequate Soil Moisture (SWT 1) and Drought (SWT 2) in Sequence throughout the Growing Period.
SWR
Flowering W1
Vegetative stages
Inflorescence formation
SWT 1
SWT 1
SWT 1
W2
SWT 2
SWT 2
SWT 2
W3
SWT 1
SWT 2
SWT 2
W4
SWT 2
SWT 1
SWT 1
W5
SWT 1
SWT 2
SWT 1
The Effect of Drought Occurring at Different Growth Stages on Productivity
of Grain Amaranth Amaranthus cruentus G6
Table 2 Plant Growth, Biomass Production and Yield Performance of A. cruentus G6 Determined at the time of Maturity in Response to Drought Initiated at different Growth Stages.
Trait ANOVA Treatments mean (cm, g, cm 2 per plant) 2
W1 W2 W3 W4 W5 Root length
SWR
47.4 b 50.2 b 50.6 b 48.4 b 63.7 a Stem height
103.8 a 65.7 c 82.1 b 104.8 a 86.7 b Inflorescence length
29.4 b 15.4 d 17.6 cd 33.3 a 19.9 c Root weight (fresh)
13.0 a 5.6 b 10.2 ab 8.5 bc 13.1 a Stem weight (fresh)
32.7 a 13.7 c 23.2 b 28.1 ab 33.3 a Inflorescence weight (f)
25.3 a 10.3 b 15.3 b 30.4 a 14.0 b Leaves weight (fresh)
18.0 ab 7.5 d 10.3 cd 19.5 a 13.8 bc Grain weight (fresh)
6.6 b 3.2 c 4.6 c 8.5 a 3.9 c Biomass (fresh)
95.6 a 40.4 c 63.6 b 95.1 a 78.1 ab Root weight (dry)
5.3 a 2.3 c 4.3 ab 2.9 c 4.0 ab Stem weight (dry)
8.8 a 4.2 b 6.9 ab 5.9 ab 8.0 a Inflorescence weight (dry)
5.6 a 2.3 c 3.4 bc 4.8 ab 4.2 ab Leaves weight (dry)
4.9 ab 3.6 c 5.3 a 4.3 ab 3.7 c Grain weight (dry)
3.9 a 1.9 c 2.8 b 4.6 a 2.5 bc Biomass (dry)
28.5 a 14.2 b 22.8 a 22.5 a 22.4 a R:S * 0.23 a 0.19 ab 0.23 a 0.15 b 0.22 a HI * 0.19 ab 0.16 b 0.16 b 0.24 a 0.14 b HI r ** 0.15 b 0.14 b 0.13 b 0.21 a 0.12 b
1 **, * Significant at the 0.01 and 0.05 probability level, respectively. 2 Means within each row followed by different letters are significantly different (Duncan, α = 0.05).
partitioning and accumulation, expressed on the fresh condition until the inflorescence formation (W4) was weight basis, was the least affected by drought
statistically the same, but in average higher than that of occurred in the period before inflorescence formation.
control (Table 2).
Dry biomass production and assimilate allocation Although, the HI obtained in the pot experiment was altered in response to water supply regimes. Plant
(Table 2) could not be extrapolated to field conditions, total dry mass decreased in case of plants exposed to
the parameter evaluated for A. cruentus G6 in field drought throughout growing cycle, while plants
trials is also relatively low and ranged between 0.19 water-stressed only for a certain periods were similar
and 0.34, with regard to growing season and date of and not differed significantly to the control. Among
sowing [9]. Those low values show the low efficiency water-stressed treatments, the reduction in assimilate
of biomass allocation into the seed of species, and allocation into the root was the most pronounced in
improvement of traits increased the harvest index is treatments W4 and W2, and similar patterns occurred
important breeding goal for this versatile and highly in respect to stem weight and root to shoot ratio (Table 2).
phenotypically plastic crop [10]. However, water In comparison to the control, grain yield of A.
regime affected both calculated harvest indexes which cruentus G6 plants growing under permanent water
values were higher in W4 treatment in comparison to stress was strongly reduced (for 51% on fresh and dry
the control (in respect to HI, the difference is weight basis). Drought stress during inflorescence
pronounced, but not significant).
formation (W3, W5) had also negative effect on grain
4. Conclusion
yield, although adequate soil moisture status in treatment W5 was restored at the beginning of
Although the grain amaranth has been considered as flowering. Grain yield of plants grown under drought
drought tolerant crop, plants exposed to water stress
286
The Effect of Drought Occurring at Different Growth Stages on Productivity
of Grain Amaranth Amaranthus cruentus G6
throughout the growing period were strongly impaired. amaranth (Amaranthus spp.) in response to soil drying, European Journal of Agronomy 16 (2002) 137-150.
Described increase in grain yield, harvest indexes and
F. Liu, H. Stützel, Biomass partitioning, specific leaf area, decrease in root to shoot ratio in treatment W4 showed
[5]
and water use efficiency of vegetable amaranth higher assimilation efficiency after plant re-watering in
(Amaranthus spp.) in response to drought stress, Scientia the period of generative growth stages. Therefore, soil
horticulturae 102 (2004) 15-27.
[6] drying in the growing period from the sowing to the E.N. Ommami, P.S. Hammes, Interactive effects of salinity and water stress on growth, leaf water relations,
beginning of inflorescence formation seems to provoke and gas exchange in amaranth (Amaranthus spp.), New and improve the assimilate allocation to the
Zealand Journal of Crop and Horticultural Science 34 above-ground biomass and particularly to the grain of
(2006) 33-44.
[7] A. cruentus G6. S.G. Mlakar, F. Bavec, Zrnati š čiri (Grain amaranth), in: A. Tajnšek, I. Šantavec (Eds.), Novi izzivi v Poljedelstvu
References (New Challenges in Field Crop Production), Slovensko
Agronomsko Društvo, Ljubljana, 2000, pp. 278-283. [1]
F. Bavec, S.G. Mlakar, Effects of soil and climatic Production and Use of Alternative Crops, CRC Press,
F. Bavec, M. Bavec, Grain Amaranths, in Organic
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conditions on emergence of grain amaranths, European Taylor and Francis Group, Boca Raton, 2006, pp.
Journal of Agronomy 17 (2002) 93-103. 88-107.
[9] S.G. Mlakar, The impact of sowing date, plant density and [2] J. Kigel, Development and ecophysiology of amaranths, in:
mineral nitrogen fertilization on grain yield and quality of O. Peredes-López (Ed.), Amaranth Biology, Chemistry
Amaranthus cruentus G6, M.Sc. Thesis, Ljubljana, 2006, and Technology, CRC Press, Boca Raton, 1994, pp. 39-75.
p. 88.
[3] L. Johnson, T.L. Henderson, Water use patterns of grain [10] D.M. Brenner, D.D. Baltensperger, P.A. Kulakow, J.W. Amaranth in the northern great plains, Agronomy Journal
Lehmann, R.L. Myers, M.M. Slabbert, et al., Genetic 94 (2002) 1437-1443.
resources and breeding of amaranthus, Plant Breeding [4]
F. Liu, H. Stützel, Leaf water relations of vegetable
Reviews 19 (2000) 229-238.
Journal of Life Sciences 6 (2012) 287-299
Structural Changes of Lignified Tissues from Sugarcane Leaves Induced by Smut (Sporisorium scitamineum) Virulence Factors
Borja Alarcón, Rocío Santiago, Carlos Vicente and María Estrella Legaz Team “Cell Interactions in Plant Symbioses”, Faculty of Biology. 2, Jose Antonio Novais Av., Madrid28040, Spain
Received: May 01, 2011 / Accepted: June 07, 2011 / Published: March 30, 2012.
Abstract: Sugarcane leaf shows the classical arrangement of cells, which defines a C 4 species. Vascular bundles consist of xylem, phloem and fibres, surrounded by an outer layer of sclereids and an inner ring of stone cells associated with the phloem. Some sclereids located below and above the vascular bundles act as docking cells and connect the vascular bundle to the internal surfaces of upper and lower layers of the epidermis. A compact mass of sclereids occupies the total internal volume of the leaf edge. Neither docking cells nor the internal mass of sclereids in the edge were markedly coloured by phloroglucinol, indicating the absence of lignin in their cell walls. However, such staining indicated that fibres of the vascular bundle and the external layer of sclereids were strongly lignified. Incubation of leaf discs with an virulence factors produced by the pathogen Sporisorium scitamineum increased the thickness of the lignified cell walls of sclereids as well as the mid and small xylem vessels, as a possible mechanical defence response to the potential entry of the pathogen. This mechanism was mainly revealed for the resistant cv. Mayari 55-14, whereas lignification decreased for the susceptible cv. B 42231.
Key words: Saccharum officinarum, Sporisorium scitamineum, fungal virulence factors, lignification, plant defence, sclereids.
1. Introduction the lower than on the upper surface of the leaf.
A cross section of a sugarcane leaf shows a
Sugarcane Saccharum officinarum L. is a C 4 plant
systematic arrangement of cells. The lower and upper whose leaves showing a typical Kranz anatomy [1-3]. epidermal layers are made up of brick-shaped cells The blade of sugarcane leaf is a long, thin, and flat with their long axes and parallel to the leaf and with structure, which gradually tapers from the base to the their cell walls thick and lignified. Bulliform cells, tip and is supported by a midrib extending practically located above or to one side of small vascular bundles, its full length. Leading off at acute angles from the often extend to the upper epidermis, thus making the midrib are numerous parallel veins, each of which epidermal layer very thin at this point. The conducting contains a vascular bundle. On the upper and lower leaf tissues or vascular bundles are found within circular surfaces are found a number of one- and two-celled and oval-shaped groups of cells. The vascular bundle trichomes, which develop from the epidermal cells includes xylem, phloem and phloem fibres, all of which lying between the veins. Abaxial epidermis showed surrounded by a ring of large cells known as bundle well-defined stomata and many regular granules or sheath cells. These, and those immediately adjoining ribbons that characterised epicuticular waxes of them on the outside of tissues, located above and below n -aldehyde nature [4]. Stomata are more numerous on each large- and medium-sized vascular bundle is a
Corresponding author: María Estrella Legaz, Ph.D., small group of long, slender but very thick-walled cells professor, research fields: analytical techniques, polyamines,
known as fibre cells. These also occur at the base of plant phathology, plant symbiosis. E-mail: [email protected].
Structural Changes of Lignified Tissues from Sugarcane Leaves Induced
by Smut (Sporisorium scitamineum) Virulence Factors
each small bundle and few are sometimes found lying above the bundle [5].
In the upper internodes, hyphal growth concludes with the formation of the whips (sori with teliospores). Teliospores are dispersed by the wind and germinate on the wet surface of sugarcane plants forming a haploid promycelium. Cells of promycelium asexually divide to form sporidia that eventually joined to form a diploid mycelium with infecting ability [6]. Hyphae do not penetrate into the cells of the scale leaves [7] and, consequently, buds tightly enclosed within scale leaves can escape infection. Entry into the bud meristem occurs between 6 and 36 h after the teliospores are deposited on the surface [8]. Hyphal growth occurs throughout the infected plant, but mostly in the parenchyma cells of the lower internodes.
Fungal hyphae have been reported to occur on the abaxial epidermis of smut-infected leaves in cv. Barbados, where penetration was obtained through open stomata. Many parenchymatous cells were invaded by smut and their cell walls were broken by actively progressing hyphae. Mucilage production accompanied the complete destruction of parenchymatous cells that harboured many fungal spores [9].
Many authors claimed about the role of the mechanical resistance of plant tissues to pathogen invasion by producing biopolymers that restrict the spread of pathogens, such as hydroxyproline-rich glycoproteins, lignin and callose [10], mainly consisting of suberized or lignified peridermis, sclereids, xylem, or phenolics in phloem and parenchymatous cells. Transgenic tobacco plants with suppressed levels of the phenylpropanoid-biosynthetic- enzyme L-phenylalanine ammonialyase and correspondingly low levels of chlorogenic acid, the major soluble leaf phenylpropanoid product, exhibit more rapid and extensive lesion development than wild-type plants after infection by the virulent fungal pathogen Cercospora nicotianae [11]. An appropriate mechanical stimulation of cucumber leaves significantly improves plant resistance and alters the activity of phenylalanine
ammonialyase leading to synthesis of lignin.
Particularly, the infection of plants by fungal pathogens produces phenols and changes the role and reaction abilities of phenolic compounds [12] as a primary response to virulence factor signalling. The early release of preformed phenolics and subsequent intensive production after stimulation of phenylpropanoid metabolism, are a part of resistance reactions to disease in many plants [13]. Within the central vacuole, the pre-existing pool of phenylpropanoids (mainly monolignols) are stored and incorporated into the cell walls following release into the cytoplasm during the initial stages of plant defence. These processes are dependent on peroxidases and other enzymes in the apoplast involved in phenolic acid esterification. Only during later pathogenesis is de novo synthesis of phenolic compounds switched on, following the transcriptional activation of genes for phenylpropanoid biosynthesis, closely associated with phenylalanine ammonialyase [14]. Lignins are formed and used to increase rigidity of cell walls, and phenolics are part of the hypersensitive response [15].
Recently, it has been found that the sensitivity or resistance of sugarcane to smut can be related to changes in the levels of free phenolic compounds, and phenylalanine ammonialyase (PAL) and peroxidase (POX) activities in the leaves. Virulence factors from S. scitamineum enhance the activity of PAL [16] and consequently increase the levels of hydroxycinnamic and hydroxybenzoic acids [17]. This effect could be directed to increase lignification, as a mechanical barrier against the pathogen, which would affect mainly a widespread population of sclereids and xylem vessels.
To verify this hypothesis, two different cultivars with different sensitivity to smut, have been chosen to study the influence of smut virulence factors, which enhances the level of phenylpropanoids, on the lignification pattern of sugarcane leaves. We propose a simple method by using light microscopy and image analysis in order to quantify the lignification degree of
Structural Changes of Lignified Tissues from Sugarcane Leaves Induced
by Smut (Sporisorium scitamineum) Virulence Factors
both sclereids and xylem.
2.3 Treatment of Sugarcane Leaves with the Smut Virulence Factors
2. Material and Methods
Twenty discs (1 cm 2 cm) were obtained from leaf
2.1 Plant Material and Culture Conditions
3 from the bottom of six different plants of both Six-month-old field grown plants of Saccharum
sugarcane cultivars (0.5 g total fresh weight officinarum (L.) cv. Barbados 42231 (susceptible to
approximately). They were floated in Petri plates on 15 smut) and Mayarí 55-14 (resistant to smut) were used
mL 10 mM phosphate buffer, pH 6.8, containing 4% throughout this study. Plants were developed from
isopropyl alcohol for 2 h at 37 °C in the dark. agamic seeds and cultured on soil in the Real Jardín
Thereafter, 0.5 mL of the virulence factors solution was Botánico Alfonso XIII (Complutense University,
added to the plates and incubated in the dark for a Madrid). Seeds were planted in April on clay soil
further 6 h [20]. Control experiments were performed in the absence of virulence factors. Three replications
mixed with 25% sand (w/w), and fertilized with
using leaf samples from different stalks were made. nitrogen (150 kg·ha ), phosphorus (75 kg·ha ) and
potassium (120 kg·ha -1 ) at planting. Plants were grown
2.4 Light Microscopy
from May to October in isolated greenhouses, under a
Each disc obtained from the central zone of the leaf light intensity of 250 µmol·m ·s of white light, a blade was cut in 10 μm thick sections using a freezer photoperiod of 14 h, a 90% relative humidity, and were microtome. Lignified structures (mainly sclerenchyma watered daily [9]. and xylem vessels) were visualized using the
-2 -1
2.2 Virulence Factors Preparation phloroglucinol/HCl (PGH) test. Sections were Teliospores of S. scitamineum (20 mg in dry weight)
incubated overnight in a solution of 1% (w/v) were isolated from whips collected from diseased
phloroglucinol in absolute methanol. Following further Barbados 42,231 plants in experimental crops of the
incubation of cleared tissues in chloral hydrate, the National Institute for Sugarcane Investigation (INCA)
sections were mounted in a few drops of concentrated in Matanzas, Cuba. The collected teliospores were
hydrochloric acid and covered with a coverslip [21]. incubated in 200 mL of sterile Lilly and Barnett
The stained sections were observed immediately using medium [18] at 38 °C for five days [19]. The mycelium
a Zeiss 60 invertoscope fitted with a CCD camera for formed was harvested, washed with distilled water,
capturing images using a Viewfinder Lite program. lightly dried with filter paper, weighed and ground to a
After 10 min, lignified structures appeared cherry fine powder in liquid nitrogen (3.6 g wet weight). The
red-orange, but colour faded within 2-4 h [22]. powder was extracted with 25 mL of 10 mM Tris-HCl,
2.5 Image Analysis
pH 8.8. Following centrifugation (5,000 g for 10 min at 4 °C), 20 mL of 80% (v/v) methanol was added to
To study the development and differentiation of the pellet and the mixture was shaken for 4 h at 38 °C.
sclerenchyma, as well as variations in thickness of the After centrifugation, the pellet was washed once with 5
cell wall of xylem elements, an image analysis was mL methanol and dried under airflow for 2 h. The dried
performed by using the program Image Tool 2.0 on pellet was washed with 10 mM phosphate buffer, pH
phloroglucinol-stained sections (the number of
6.8, then resuspended in 25 mL of the extraction buffer, replicates was done in the corresponding table). autoclaved (120 °C for 30 min) and re-centrifuged [16].
For the study of the area occupied by lignified cells The clear supernatant was used to elicit the sugarcane
in a cross section of leaf blade, two zones of reference leaves.
were arbitrarily chosen. The first zone includes the
Structural Changes of Lignified Tissues from Sugarcane Leaves Induced
by Smut (Sporisorium scitamineum) Virulence Factors
epidermal cells that contact with docking cells
3. Results
(subepidermal sclereids that connect with the
3.1 Light Microscopy of Sugarcane Leaves conductive bundles), longitudinal plates or strands of
hypodermal sclerenchyma associated to almost all of Cross-sections of sugarcane leaves from B 42231 the longitudinal bundles of the blade [23], as well as the
and My 55-14 shows the classical arrangement of cells, proper docking cells, and the vascular bundle, including
which defines a C 4 species. Fig. 1 only shows this bundle sheath cells. The second zone includes arrangement for My 55-14 leaves since B 42231 exclusively vascular tissues and associated sclereids.
exhibits identical structure at the level of light To analyze the thickness of the sclereid cell wall in
microscopy. On the left, appeared control leaves and treated and control leaf discs, three areas were
the right hand, those treated with the virulence factors. measured in each cell: the total area of the cell, the area
On the upper and lower leaf surfaces are found a of the cell lumen and, for difference between both, the
number of one- and two-celled trichomes, which area of the cell wall. Then, the percentage of the cell
develop from the epidermal cells lying between the surface that represents the lignified cell wall of
veins. Inside the edges of leaves, having sharp marginal sclereids was calculated for control leaf discs and for
teethes inclined toward the apex of the leaf, a dense those treated with the virulence factors. An identical
mass of sclereids occurs below the epidermal layer procedure was used to study variations in the thickness
(Figs. 1A and 1B). Bulliform cells occur in the lamina of S. officinarum . These thin-walled cells of this
of the cell wall in the xylem elements of treated and monocotyledonous plant, the size of which is similar to
control leaf discs. that of brick-shaped epidermal cells, are confined to the
2.6 Statistics
Statistical significance of the differences between means of the analyzed parameters was evaluated by student’s t-test. The f-test was used to test heterogeneity of variances. Differences were considered significant when P 0.05.
2.7 Scanning Electron Microscopy For ultrastructural studies, leaves 3 and 4 from the
bottom of six different plants, at a middle position on the stalks, were always chosen. The ultrastructure of sugarcane leaves was examined by conventional scanning electron microscopy (SEM). Samples were
Fig. 1 Light micrographs of cross-sections of leaves of
fixed in 2% glutaraldehyde (v/v) in 0.1 M phosphate
sugarcane, cv. My 55-14 without any treatment (A y C) and
buffer, pH 7.2, post-fixed with osmium tetroxide,
treated with crude virulence factors (B y D), stained with
washed, dehydrated in acetone, critical-point dried,
phloroglucinol/HCl. (A and B): Leaf edge showing adaxial (UE)
sputter-coated with gold/palladium and scanned at and lower (LE) epidermis, a mass of sclereids (S) under
epidermis and mesophyll cells (MC). (C and D) Magnification
20 kV by SEM [24] using a JEOL JSM 6400 (Japan).
of the vascular bundle showing both adaxial (UE) and abaxial
Digital images were obtained by using an INCA
(LE) epidermis, large (lXy), middle (mXy) and small (sXy) (Oxford) program incorporated to the equipment xylem, phloem (Ph), stone cells (SC), docking cells (DC),
[9]. bundle-sheath cells (BS), and mesophyll (MC).
Structural Changes of Lignified Tissues from Sugarcane Leaves Induced by Smut (Sporisorium scitamineum) Virulence Factors
adaxial epidermis (not shown). The conducting tissues are found within the circular to oval-shaped groups of cells. The bundles are composed of three-sized classes of cells: large, medium and small, the first two being rhomboid to oval in shape, while as a rule, the small type is rather circular. A small, round bundle always lay next to a large vascular bundle, which usually extends from the upper to the lower epidermis of the leaf (Figs. 1C and 1D). The xylem is made up of open tubes or vessels associated with smaller and thicker walled elements. The large bundles of the leaf are usually two large vessels connected with smaller vessels. The xylem of small bundles consists of only a few large pitted vessels. The large vessels were irregular in shape, having comparatively thick walls (Figs. 1C and 1D).
Each vessel is formed not from a single cell but from
a series of elongated cells, whose contents and end walls have disappeared. According to Martin [5], the vascular bundle includes phloem fibres (called stone cells), the function of which, due to their structure and arrangement, is mainly to give strength to the leaf. They are long, slender, pitted cells with pointed ends and with thick walls which are lignified. Their cell cavities are extremely small. These fibre cells may occur singly but they are usually in groups forming strands, which extend longitudinally. Sclereids also are located below and above the vascular bundle, connecting it to the internal surface of the epidermal, upper and lower layers. They are docking cells that immobilized and ensured the total vascular bundle inside the spatial structure of the leaf. De visu observation of these micrographs seems to reveal a higher degree of colour intensity of the sclereids and vessels from those leaves treated with virulence factors than in untreated leaves. However, this appreciation needs to be confirmed.
As it can be seen in Fig. 1, the edge of the My 55-14 growing leaf showed a ring that corresponded to the cross section of a spicule. The reason for it to appear in these micrographs and not in those of the cultivar B 42231 is only casual (data not shown). Fig. 2 shows the
mean number of spikes of each cultivar per cm 2 . There was not significant differences between treated or untreated leaves neither from B 42231 cv. nor from My 55-14 cv.. However, although differences between cultivars were not significant, their number in the resistant cultivar is higher than that of the sensitive one.
3.2 Image Analysis of Lignified Tissues In order to quantify the degree of lignification, it has
been performed an image analysis from micrographs. Results from these analysis will be shown in Tables 1-3. Table 1 shows the results of the image analysis designed to assess changes in the pattern of lignification, performed on leaf sections stained with phloroglucinol that reveals the lignified structures. The results of the area measurements were divided in different sections, firstly, differences found according to the sugarcane cultivar, Barbados 42231 or Mayari 55-14; after this, these obtained according to the used treatment, control (untreated) or leaf discs incubated with a solution of virulence factors (with treatment), and finally, as the absolute size of the total area of the vascular pockets, small and large. Absolute measurements are shown in area counts, the percentage relative to total area of reference and the percentage relative to the area of the vascular bundle. The results marked with asterisk have a P ≤ 0.05 and they were considered as statistically significant.
It can be observed in Table 1 that the area of the
B 42231 C B 42231 T M y 55-14 C My 55-14 T
Fig. 2 Spicule number per cm 2 quantified in cross-sections of sugarcane leaves of susceptible (B 42231) and resistant (My 55-14) cultivars, treated (T) or not (C) with the crude virulence factors.
Structural Changes of Lignified Tissues from Sugarcane Leaves Induced
by Smut (Sporisorium scitamineum) Virulence Factors
Table 1 Area of Sclereid Groups in Cross-sections of Sugacane Leaf Blade from Barbados 42231 and Mayari 55-14 Cultivars, Treated or not with Virulence factors from Sporisorium scitamineum. Image analysis was Performed by using the Image Tool 2.0 Program. Values are the mean of 30 different Replicates ± Standard Error. * Indicates Values from Treated-cross-sections that differ from the Corresponding Untreated Leaves in P ≤ 0.05.
Area measurements (arbitrary area counts) Without treatment
With treatment Bundle size
Cell type
Area counts
% of the total selected area
Area counts % of the total selected area
Adaxial docking cells
Abaxial docking cells
Total vascular bundle
Sclereids from vascular bundle
Total selected area
Adaxial docking cells
Abaxial docking cells
Total vascular bundle
Sclereids from vascular bundle
Total selected area
Adaxial docking cells
Abaxial docking cells
Total vascular bundle
Sclereids from vascular bundle
941,701 100 Mayari 55-14
Total selected area
Adaxial docking cells
Abaxial docking cells
Total vascular bundle
Sclereids from vascular bundle
Total selected area
Table 2 Percent of Sclereids Cell area Corresponding to the Cell wall in Cross-section of Sugarcane Leaves from Barbados 42231 and Mayarí 55-14 cv. Treated or not with Virulence Factors from Sporisorium scitamineum. Image analysis was Performed by using the Image tool 2.0 Program. Values are the mean of 30 different Replicates ± standard error. * indicates Values from Treated-cross-sections that differ from the Corresponding Untreated leaves in P ≤ 0.05.
Area measurements (arbitrary area counts) Without treatment
With treatment Cultivar Cell type
Cell
Cell wall [Cell wall/Cell] Cell
Cell wall [Cell wall/Cell]
area 100 Sclereids in the leaf edge
6,263 4,836 77.20 ± 1.29 Adaxial docking cells
Abaxial docking cells
6,784 6,178 91.06 ± 0.75 Sclereids surrounding xylem
Stone cells
11,063 8,019 72.49 ± 1.34* Sclereids surrounding the vascular bundle 7,750 6,576
8,923 7,297 81.77 ± 1.43 Sclereids in the leaf edge
7,712 6,419 83.24 ± 0.85* Adaxial docking cells
Abaxial docking cells
5,841 5,582 95.56 ± 0.45* Sclereids surrounding xylem
Stone cells
8,603 6,769 78.68 ± 1.06 Sclereids surrounding the vascular bundle 9,873 8,225
7,222 6,292 87.12 ± 1.17 ** Cell wall area = cell area – lumen area.
Structural Changes of Lignified Tissues from Sugarcane Leaves Induced
by Smut (Sporisorium scitamineum) Virulence Factors
Table 3 Percent of Xylem Vessels Cell area Corresponding to the Cell wall in Cross-section of Sugarcane Leaves from Barbados 42231 and Mayarí 55-14 cv. Treated or not with Virulence Factors from Sporisorium Scitamineum. Image Analysis was Performed by using the Image tool 2.0 Program. Values are the mean of 30 Different Replicates ± standard Error. * Indicates Values from Treated-cross-sections that Differ from the Corresponding Untreated Leaves in P ≤ 0.05.
Area measurements (arbitrary area counts)
Cultivar Bundle size
Without treatment
With treatment
Cell area Cell wall area [Cell wall/Cell] 100 Large
Cell area Cell wall area [Cell wall/Cell] × 100
18.98 ± 0.53* Barbados 42231 Middle
24.27 ± 0.70 Mayari 55-14
sclereids surrounding the vascular bundle and inside vascular bundle was similar in both cultivars, while the the vascular bundle itself relative to total area of
small vascular bundle was lower in My 55-14. The area reference in the growing susceptible B 42231 suffered
occupied by sclereids surrounding large vascular bundle
a significant decrease after the elicitation treatment, was significantly higher in the susceptible cultivar, but both in large as well as in small vascular bundles. The
after treatment this difference was reversed. area occupied by docking cells decreases significantly,
Table 2 shows the absolute measurements obtained affected only after elicitation treatment in the case of
after the analysis with the Image Tool 3.0, as well as small vascular bundles. The area occupied by these
the percentage of cell area of the sclereids which cells in large vascular bundles was not significantly
represented the area cell wall with respect to total area altered after incubation with the virulence factors
of the cell in the leaf cuttings from two cultivars of solution. For the My resistant cultivar after treatment, a
sugarcane (B 42231 and My 55-14), control and treated significant increase in area occupied by sclereids of the
with extract of crude virulence factors. The results vascular bundle was achieved, most evident for the
marked with asterisk were considered as significant. small but also statistically significant in the large
In the cultivar B 42231, there was a significant vascular bundles. The area occupied by docking cells
decrease in the cell wall thickness of the spring cells on the adaxial epidermal layer and the percentage area
(especially those of the adaxial side) and sclereids of sclereids in the area of the small vascular bundles
surrounding the xylem, while the thickness of the wall were the only results that deviate from this general trend,
of the other types of sclereids does not significantly which suffered a significant decrease after treatment
change after treatment. However, for the cultivar My with extract of crude virulence factors. In general, one
55-14 was observed the contrary to what happens in could say that after incubation, lignification areas
cultivar B 42231, i.e., an increased wall thickness of decreased for most of the sclereids in the susceptible
the spring cells of both the adaxial and abaxial layers, cultivar, while increased in the resistant cultivar.
as well as edge of the leaf cells after treatment with After analyzing the percentage of area occupied by
crude virulence factors. B 42231 latter remained each type of sclereids in both cultivars, it could be seen
constant. The variation of wall thickness was not that the rugged area occupied by both adaxial and
significant, the confidence level for the variable tax on abaxial docking cells was greater in resistant than in the
the rest of the different types of sclereids. Comparing susceptible cultivar. This morphology, which seems to
the two cultivars, there was no difference between the
be of varietal origin, was not changed after treatment thickness of the wall of the sclereids from any group with the crude virulence factors. The large size of
studied. However, the wall thickness of the sclereids
Structural Changes of Lignified Tissues from Sugarcane Leaves Induced
by Smut (Sporisorium scitamineum) Virulence Factors
from the resistant cultivar showed to be greater than the clearly seen, which were only isodiametric in cross wall thickness of the sclereids from the susceptible
section as they are fibres. Xylem vessels also differed cultivar, after treatment with a solution of virulence
in the medium size. In the centre of Fig. 3D, it appears a factors. The woody structure corresponded to the cell
xylem medium vessel, strongly lignified, surrounded wall of stone cells.
by small xylem vessels whose walls were thinner. It Table 3 shows the absolute measurements obtained
can also be appreciated the so-called spiral ribs at the after the analysis with the Image Tool 3.0, as well as
internal surface of the vessel.
the rate of the cell wall of xylem elements large,
4. Discussion
medium and small leaf sections of two sugarcane cultivars sugar (B 42231 and My 55-14), control
Saccharum officinarum L. presents a Kranz anatomy (without any treatment) and treated with the virulence
typical of plants with C 4 photosynthesis, leaf structure factors solution. The results marked with asterisk (P
that is markedly different from that C 3 plants. In the be
0.05) were considered as significant. The cell wall of most simplified way, Kranz anatomy of C 4 plants can xylem with elements tended to thicken after incubation
reduced to the existence of a vascular sheath with a solution of virulence factors in both cultivars.
surrounding the conducting bundles, formed by large The significant differences occurred in the case of B
cells with agranal chloroplasts, surrounded by a layer 42231, where drivers do large and small, in the case of
of mesophyll composed of cells radially arranged to My 55-14, in the medium and small, although the largest
form chlorophyllous crown [25]. increases always occurred in the resistant cultivar.
In leaf cross sections (Figs. 1C, 1D, and 3A), the leaf Varietal xylem vessel walls My 55-14 was
blade exhibits the characteristic Kranz anatomy of C 4 significantly thicker than the xylem vessels of B 42231,
plants [26] with sheath cells radially arranged around and this relationship remained after the elicitation
the vascular bundles. These sheath cells or true Kranz treatment. It can observe a direct correlation between
cells are characterized by cell walls thicker than those xylem vessel size and degree of lignification, as the
in the mesophyll cells, with numerous pits and cell latter increased with decreasing the size of conductors.
walls traversed by plasmodesmata that connect the protoplasm of both cell types. These cells are located so
3.3 Ultrastructure of Lignified Tissues dense and compact drawn virtual intercellular spaces.
Fig. 3A shows two vascular bundles, one large and Their number is more limited than that of mesophyll one small from untreated B 42231 leaves. It can be
cells, in the order of one fifth in the sugar cane. appreciated bulliforms cells, adaxial and abaxial
Mesophyll cells, are establishing contacts with each epidermis, sheath cells, phloem and large, medium and
other, leaving large intercellular spaces between them small xylem vessels. Mesophyll was observed between
[27]. In the vascular bundle, xylem elements can be the vascular bundles. This structure corresponded to
distinguished as large, medium and small xylem vessels
and phloem. Most of the vascular bundles are the detail of a vascular bundle seen in the adaxial
the typical Kranz anatomy of C 4 plants. Fig. 3B shows
associated with large amounts of sclerenchyma, epidermis, two large xylem vessels, a xylem vessel
particularly in the abaxial surface of the leaf, as stone medium, several small xylem vessels, stone cells
cells. Mesophyll cells have subepidermal sclereid surrounding phloem vessels that contribute to cell
beams that connect vessels and whose role is to cushion integrity and docking cells that connected the vascular
the vascular bundle deflections that the wind can exert bundles below the adaxial epidermis. In Fig. 3C, stone
on the conducting elements, and appear in both abaxial cells from treated leaves of My 55-14 leaves can be
and adaxial surfaces. Fig. 3A shows bulliform cells,
Structural Changes of Lignified Tissues from Sugarcane Leaves Induced
by Smut (Sporisorium scitamineum) Virulence Factors
Fig. 3 SEM micrographs of cross-section of sugarcane leaves. (A): Ultrastructure of two vascular bundles, different in size, from untreated B 42231 cultivar. (B): Xylem vessels from cv. B 42231 treated with crude virulence factors. (C): Two stone cells from cv. My 55-14 treated with the virulence factors. (D): Fine ultrastructure of a mid vessel from the cv. Mayari 55-14 showing spiral ribs. lVB = large vascular bundle; sVB = small vascular bundle; BC = bulliform cells; UE = adaxial epidermis; LE = lower epidermis; BS = bundle-sheath cells; Ph = phloem; lXy = large, mXy = middle, and sXy = small xylem vessels; MC = mesophyll cells; SC = stone cells; DC = docking cells, R = spiral ribs. Mesophyll (M) was observed between the vascular bundles. This structure corresponded to
the typical Kranz anatomy of C 4 plants.
thin-walled cells similar in size to large xylem elements In the present study, the stain phloroglucinol/HCl that contribute to leaf-roll in water stress conditions.
gives negative results for the walls of phloem elements In the leaf edge, both treated and untreated My 55-14
and reacts positively to the secondary walls of tracheids. leaves (Figs. 1A and 1B) show a dense mass of
According to Evert et al. [28], the thin wall of the sieve sclereids in the epidermis, occupying the whole surface.
tubes is not lignified.
They are cells that maintain the linear filling of blade The image analysis designed to assess changes in the edge, increasing the mechanical strength. Micrographs
pattern of lignification has been made on leaf sections of the growing leaf edge of My 55-14 show empty
from both cultivars B 42231 and My 55-14, stained spaces corresponding to the insertion of each spike.
with phloroglucinol to reveal lignified structures (Fig. While the differences are not significant between
1). The ring of sclereids surrounding the vascular control and treated leaves, the number of spicules per
bundle, the stone cells associated with phloem and cm 2 (Fig. 2) is higher in the resistant cultivar than in the
sclereids outer leaf edge of the landfill are stained in sensitive. This could be interpreted as a further increase
red with phologlucinol/HCl. This fact is not true for the in the mechanical strength against the attack or even
epidermis, the sheath cells and sclereids innermost from certain animals.
edge, because they have no secondary wall which The conductive elements can be of three types
specifically is stained with phloroglucinol/HCl. De according to their size and structure: large, medium and
visu analysis of these micrographs reveals a more small [23]. Although our analysis only differentiated
positive reaction with phloroglucinol/HCl in those between large and small, no significant differences
leaves treated with extract of the virulence factors were found to separate into three types.
solution than in control leaves of the resistant cultivar
Structural Changes of Lignified Tissues from Sugarcane Leaves Induced
by Smut (Sporisorium scitamineum) Virulence Factors
(Fig. 1), but this does not occur in the susceptible cv. It might say that the increase of lignification of leaf (data not shown). The image analysis has been used to
sclerenchyma in the resistant cultivar is a mechanical quantify these changes in the pattern of lignification.
response of plant defence against the attack of S. In the blade cuts of the susceptible cultivar, B 42231,
scitamineum , which is a fact that does not happen in the previously treated with the virulence factors solution, a
cultivar susceptile to smut disease. significant decrease in the value of cell area occupied
From these experimental facts, it can be concluded by mesophyll, both adaxial and abaxial young bundle
that the activation of a delignification response in B vessels as well as sclereids of vascular bundle and the
42231 mainly affects to sclereids (Table 2). This would package itself with respect to the results obtained
not only arrest of lignification, but also a quantitative without any treatment (Table 1). It can be observed that,
loss more or less large of previously deposited lignin. after treatment, a significant decrease in wall thickness
In the absence of the pathogen, but in the presence of of the mesophyll cells is produced, especially on the
crude virulence factors isolated from the mycelium of adaxial as well as the sclereids surrounding the xylem
the same pathogen, a hypothesis to be verified would and the vascular bundle when compared to control
be the induction (or activation) of a complex leaves without any treatment (Table 2). The cell wall of
glycosylated lignin peroxidase, capable of being xylem elements tends to thicken, especially in large
secreted by producing cells, which moves freely in the and small vessels (Table 3).
intercellular spaces to achieve the sclereids [29] where In leaf cuttings of the resistant cultivar, My 55-14,
they carried out the oxidative breakdown of the treated with an extract of a solution of virulence factors
polymer. The increase in peroxidase activity should be is detected an inverse behaviour to that obtained in the
associated to an increase of peroxidase activity capable susceptible cultivar. The value of cell area occupied by
of generating exocellular H 2 O 2 using GSH or NADPH 2 the docking sclereids between the abaxial surface and
[30] or more commonly a fungal glucose-oxidase [31]. the vascular bundle and vascular bundle itself increases
A similar system has been described for radish cell significantly after treatment compared with control
walls [32] and xylem cell walls isolated from Forsythia sheets, especially in young conducting elements (Table
[33, 34]. The hydrogen peroxide formed from 1). In parallel, there is an increase in wall thickness that
superoxide radical (O •- 2 ) generated by reduction of O 2 produces a reduction of their lumen. This happens for • with NAD radical, a product of the oxidation of
both docking cells and the sclereids inside the foliar NADH coupled to the conversion of malate to edge. This would imply that the vascular bundles 2+ oxaloacetate. The peroxidase-Mn required phenols to
would be more anchored to the inner surface of the
be oxidized and to produce O •- 2 . Lignin is formed via epidermis, increasing its resistance to environmental
oxidative polymerization of monolignols within the factors that shake, roll or fold the leaves, even against a
cell wall matrix [35].
fungal mycelium that potentially tries to invade those Lignin peroxidases are ubiquitous cell wall enzymes packages. In this case, the treatment does not cause an
that appear to be involved in oxidative polymerization increase in wall thickness in the sclereids surrounding
of monolignols. In recent years, it has been described the xylem or vascular bundle as a whole, or of the stone
that not only laccases but also secreted peroxidases in cells associated to the phloem (Table 2). The complete
the secondary walls are able to polymerize vascular bundles and phloem would not constrained by
monolignols in the presence of O 2 . Bao et al. [36] the increase in volume of these cells that embrace. The
found that laccase activity associated to the cell wall cell wall of xylem medium and small items also increased
correlated with lignification in loblolly pine xylem, and after treatment in the resistant cultivar (Table 3).
described that it was capable of oxidizing monolignols.
Structural Changes of Lignified Tissues from Sugarcane Leaves Induced
by Smut (Sporisorium scitamineum) Virulence Factors
The laccase activity is not only involved in acids, precursors of monolignols, notably syringic lignifications processes but also in delignification,
caffeic acid linked to the cell wall [17] justify this although it has been studied more extensively in fungi,
Phoenix dactylifera callus also there are some references in higher plants [37].
conclusion.
accumulates phenolic compounds in response to the Laccases as well as p-diphenol: dioxygen reductases
elicitation with fungal culture filtrates of Fusarium are part of a large group called multicopper enzymes.
f. sp. albenidis [44]. These phenols are They can be divided into two main groups with clear
oxysporum
mainly hydroxycinnamic acid derivatives that would differences as a function of their origin, the vascular
be the main compounds involved in the resistance of plants and fungi [38]. The enzymes are able to degrade
the palm vascular disease. It could then be considered lignin in the absence of lignin peroxidase and
that accumulation of caffeic acid, described by manganese peroxidase. The molecule of laccase is a
Santiago et al. [17], in growing leaves of My 55-14 glycoprotein that occurs as a dimer or a tetramer and
after elicitation, would be on the basis of the resistance usually contains four copper atoms per monomer,
mechanism of this plant to smut.
distributed in three redox sites [39]. The enzyme Cell cultures of Capsicum annuum L. respond to the catalyzes the oxidation of ortho-diphenols and elicitation with mycelium or filtrate lyophilized fungus aminophenols, polyphenols, polyamines, lignins and
Phytophthora capsici increasing the level of aryl diamines [40]. It has been characterized in Rhus
peroxidase activity and de novo expression of acidic vernicifera , Aesculus parviflora leaves and green stems
peroxidase isoenzymes in cell wall fractions [45]. The of tea [41]. All the members of the Anacardiaceae
action of biosynthetic peroxidases and laccases family have laccase in resin ducts and resin itself [42].
required for the polymerization of monolignols in My Cell cultures of Acer pseudoplatanus L. are able to
55-14 should be associated to the cell walls of the secrete to the medium, regardless of cell growth,
sclereids and vessels that will be reinforced, in contrast laccase which represents 2% of total proteins to running for the leaves of cultivar B 42231. This synthesized by cells in the exponential phase of growth.
location in wall has been demonstrated for a system of The enzyme is similar to that described in R.
P. taeda [36] during the process of lignification. These vernicifera but differs from that of fungi [43].
enzymes are later secreted to some distance of the cells If the existence of these exocellular laccases was
that produce them.
confirmed in B 42231, as response to the action of The lignification response observed for My 55-14 virulence factors, delignification of large sclereids
leaves can seem very short (48 h). However, cotton observed in Table 2 could be explained by this
hypocotyls elicited with lipopolysaccharide isolated enzymatic action. Its molecular mass of approximately
from Verticillium dahliaea produce increased 100 kDa would not be a problem for the mobility of the
lignification, visible only after six hours after enzyme and its glycosylation in the Golgi system
elicitation and reached a peak at 22 h [46]. This would link up with its internalization in secretory
elicitation is logically associated to an increased PAL vacuoles to be removed from the cell [43].
and cell wall-peroxidase activities. The cell wall glucan By contrast, the increase in the pattern of
of Phytophthora nicotiana response elicits massive lignification observed in the resistant cultivar, My
accumulation of phenolic polymers in cotyledons of 55-14, would be clearly related to the increased activity
Glycine max peaking at 24 h [47]. It would be of PAL and peroxidase after elicitation. This has been
interesting to note that the laccase of A. pseudoplatanus demonstrated recently [20]. Nevertheless, the increase
monolignols are able to polymerize in the complete in the levels of hydroxybenzoic and hydroxycinnamic
absence of peroxidase [48]. This could be interpreted
Structural Changes of Lignified Tissues from Sugarcane Leaves Induced
by Smut (Sporisorium scitamineum) Virulence Factors
as meaning that laccases may be involved only in the establishment in the host and production of whips in sugarcane smut (Ustilago scitaminea) of sugarcane, in:
early stages of lignifications, while peroxidases act as Proceedings of the International Society of Sugarcane
catalysts for posterior polymerization. Technolology,1980, pp. 1452-1455. Since the levels of accumulation of hydroxycinnamic
[9] M.E. Legaz, A.M. Millanes, B. Fontaniella, D. Piñón, R. and hydroxybenzoic derivatives in leaf segments from
De Armas, C.W. Rodríguez, et al., Ultrastructural alterations of sugarcane leaves caused by common
My 42231 and B 55-14 after elicitation are different, and sugarcane pathogens, Belgian Journal of Botany 139
higher in the resistant cultivar than in the susceptible [17,
(2006) 14-26.
20]. It could be considered as hypotheses for future [10] J. Kuc, Compounds from plants that regulate or participate work not only the induction of laccases and peroxidises, in disease resistance, Ciba Foundation Symposia 154
(1990) 213-224.
E.A. Maher, N.J. Bate, W. Ni, Y. Elkind, R.A. Dixon, C.J. proposed by Eggert et al. [49]. According to these
but a fine control of the system, similar to which
Lamb, Increased disease susceptibility of transgenic authors, the level of phenols (or one of them in
tobacco plants with suppressed levels of preformed phenylpropanoid products, in: Proceedings of the National
particular) would displace the equilibrium towards Academy of Sciences, 1994, pp. 7802-7806.
depolymerizing oxidative reactions, which would be
D. Sedlá řová,, A. Lebeda, Histochemical detection and interpreted as a response of susceptibility, or of
role of phenolic compounds in the defense response of Lactuca resistance when the equilibrium displaces to oxidative spp. to lettuce downy mildew (Bremia lactucae), Journal of Phytopathology 149 (2001) 693-697. polymerization. The ability of laccases to degrade
[13] S. Peltonen, Responses of barley and wheat to pathogens, lignin increases if certain phenols act as mediators.
nonpathogens and wounding as indicated by induced phenylalanine ammonia-lyase activity, Acta Agriculturae
Acknowledgments
Scandinavica B. Soil and Plant Sciences 48 (1998) 184-191.
This work was supported by a grant from the [14] L. Guidi, E. Degl´Innocenti, S. Genovesi, G.F. Soldatini, Ministerio de Ciencia y Innovación (Spain), BFU Photosynthetic process and activities of enzymes involved in the phenylpropanoid pathway in resistant and sensitive
2009-11983. genotypes of Lycopersicon esculentum L. exposed to ozone, Plant Science 168 (2005) 153-160.
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Journal of Life Sciences 6 (2012) 300-303
The Contribution of the Dromedary in the Spontaneous Plant Seeds Transfer in the Northern Algerian Sahara
1 1 1 Trabelsi Hafida 2 , Senoussi Abdelhakim , Chehma Abdelmadjid and Faye Bernard 1. Laboratoire de Bio-ressources Sahariennes, Préservation et Valorisation, Université Kasdi Merbah, Ouargla 30000, Algérie
2. CIRAD-ES, Montpellier Cedex 34398, France
Received: July 23, 2011 / Accepted: August 18, 2011 / Published: March 30, 2012.
Abstract: The current study focuses on the contribution of the dromedary in the preservation and increase of spontaneous flora through seeds transfer by endozoochory. Thus dromedary faeces have been collected in selected area of region of Ghardaia (Northern Algeria Sahara), since it is one of the three known cameline rangelands during the four successive seasons of the years 2009-2010, in order to examine their seed contents. Our investigation allowed us to invento 1,832 seeds representing 33 different types varying according to the seasons of harvest. The biggest density of seeds was present in summer with 986 seeds, against 424 seeds in winter, 366 seeds in fall and 56 seeds in spring, and of the same way, the biggest number of types was present in summer with 30 types, consistent of the fall season with 26 types, and arrived then the season of winter with 20 types and the one of spring with five types. The gotten results allowed us to appreciate the ecological role of this animal in the desertic ecosystem in the dissemination and the proliferation of the seeds of the spontaneous flora in its fragile and hostile desert environment to the survival of the seeds enveloped in its faeces.
Key words: Dromedary, seeds, endozoochory, Saharan rangelands, Ghardaïa.
1. Introduction
camels in the region of Ghardaia, during four seasons of the years 2009-2010.
In Sahara, desert plants are represented by a small number of taxa in relation to the available area, which
2. Experiment
might suggest that their power of colonization is low,
2.1 Harvest Faeces
however, they can spread over vast distances, seed dispersal is facilitated by wind and also by the
The samples of faeces were held on area of migration of humans and animals [1]. Apart from the
Ghardaia representing three types of rangelands spread of seeds by wind, the spread caused by man and
(Wadi bed, Depression, Hamada) and the four seasons animals can be considered relatively less important, in
each of the year 2009-2010. In this area, we selected view of this vast Sahara ecosystem (human poverty and
three sites relatively distant one of the other as a very low component fauna).
rational choice based on the observation of abundant In the Sahara ecosystem, the camel is the main farm
herds of camels.
animal using plant resources and can contribute to the
2.2 Seed Collection
transfer of seeds per endozoochory [2, 3]. This is in view to highlight the quantitative importance of seeds
To highlight the seeds in the faeces of camel, a transferred by the camel that fits the work, based on the
quantity of 50 g of dung randomly selected samples analysis of faeces collected in three types of rangelands
were cleaned, weighed and peeled manually using a dissecting microscope (magnification 10 × 1.8), for
Corresponding author: Trabelsi Hafida, Ph.D. candidate, research fields: ecology and environment. E-mail: collecting seeds. The collected seeds were selected, [email protected].
The Contribution of the Dromedary in the Spontaneous
Plant Seeds Transfer in the Northern Algerian Sahara
counted and encoded into several types according to while during the spring season is recorded 56 seeds their morphology, size and color, before they are stored
(Fig. 1).
in sealed vials. Seasonal variability in the number of identified seeds is directly related to the temporal variability in
3. Results and Discussions
production of plant biomass grazed rangeland. Indeed,
3.1 Identification of Seeds the work of Chehma [4] showed that the phytomass The total number of seeds identified in the faeces of
varies from season to season and that better values are the camel is of 1,832 seeds distributed in a manner
recorded in the spring. To this end, the high density of different spatiotemporal. Morphological differences
seeds in summer is a logical continuation of the (shape, size and color) of the seeds identified are
availability of plants at flower stage in spring and seed grouped into 33 different types, assumed to represent
stage to the next season.
33 different species (Table 1).
3.3 Types of Seeds
3.2 Total Number of Seeds Identified The number of identified 33 types is unevenly
The summer season includes the largest number with distributed by season. Indeed, the summer season is the 986 seeds, two times more than other seasons, followed
most represented with 30 types, followed by the fall by winter and fall, respectively with 424 and 366 seeds,
season with 26 types, and then comes the winter season
Table 1 Seeds inventoried in the faeces of the dromedary.
Season of harvest Types of rangelands Site of harvest Number of seeds Number of types Total of seeds Total of types Wadi bad
S1
Spring 2009 Depression
33 1 56 5 Hamada S3 1 1 Wadi bad
Summer 2009 Depression
63 11 986 30 Hamada S3 14 4 Wadi bad
Fall 2009 Depression
22 8 366 26 Hamada S3 280 12 Wadi bad
Winter 2010 Depression
10 424 20 Hamada S3 61 6
S2
Fig. 1 Distribution of the number of seeds according to the seasons.
The Contribution of the Dromedary in the Spontaneous
Plant Seeds Transfer in the Northern Algerian Sahara
Fig. 2 Distribution of types of seeds according to the seasons.
with 20 types and the spring season with five types (Fig. and dispersed in the environment provide favorable 2).
conditions for the preservation and germination The predominance of types of seeds in summer
of seeds.
shows, first, the adult stage of species grazed and From this we can assume that the dromedary helps availability of fruit, in fact, according to Correra [5],
preserving its environment and its role in seed dispersal, Saharan plants begin to develop their aerial part at the
which can be of great ecological importance in the end of the winter season (period of more rain) and
community extremely fragile.
continue through the spring and peak in early summer.
Acknowledgments
Second, phenological phase through the plants during this season, in fact, research shows that most plants
The present work is the basis of the project CMEP bloom in spring Sahara [6-8], so, the general
TASSILI n 09 mdu 754 on the impact of raising maturation of seeds coincides with the summer.
camelin on the environment of the northern Algerian Conversely, the autumn is characterized by the
Sahara.
breakdown and the liberation of seeds, resulting the
References
decline of the number and types of seeds followed by winter which represents the start of vegetative stage of
[1] P. Ozenda, Flore et végétation du Sahara, Centre National the majority of plants. de la Recherche Scientifique (CNRS), Paris, 1983, p. 662.
(in French)
4. Conclusion [2] P.H. Gauthier, Contribution à l'étude de l'écophysiologie
du dromadaire en été dans son milieu naturel (Moyen et Our results show the particular role of the camel as
haute Maurotanie), Extrait du bulletin de l’I.F.A.N., série
a vector of seed dispersal quantitatively and A. n 2, 1977. (in French) [3] A. Chehma, B. Faye, D. Bastianelli, Valeurs
qualitatively, in spite of the physiological nutritionnelles des plantes vivaces des parcours sahariens characteristics and potential gastrointestinal known
algériens pour dromadaires, Fourrages 204 (2010) for their efficiency to digest the walls including lignin,
263-268. (in French)
which should in principle attack the seed coat and [4] A. Chehma, Etude floristique et nutritive des parcours camelin du Sahara septentrional algérien Cas des régions
their organic content. de Ouargla et Ghardaïa, Thèse Doctorat, Université Badji In addition, the faeces in which the seeds are stored
Mokhtar, Annaba, 2005, p. 178. (in French)
The Contribution of the Dromedary in the Spontaneous
303
Plant Seeds Transfer in the Northern Algerian Sahara
[5] A. Correra, Dynamique de l’utilisation des ressources
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fourragères par les dromadaires des pasteurs nomades du [7] P. Quezel, S. Santa, Nouvelle flore de l'Algérie et des parc national du Banc d’Arguin (Mauritanie), Thèse
régions désertiques méridionales, Tome 2, 7eme édition, Doctorat, Museum National d’Histoire Naturelle de Paris
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Paris, 1963, p. 1170.
[6] A. Chehma, M.R. Djebar, F. Hadjaiji, L. Rouabeh, Etude [8] A. Chehma, Catalogue des plantes Spontanées du Sahara floristique spatio-temporelle des parcours sahariens du
septentrional algérien, Dar el Houda (Ain Mlila. Algérie), sud-est algérien, Sécheresse 16 (2005) 275-285. (in
2006, p. 156.
Journal of Life Sciences 6 (2012) 304-311
Biological Status of Captive Crane in Southern Districts of Northern Pakistan
Farzana Perveen Department of Zoology, Hazara University, Garden Campus, Mansehra 21300, Northern Pakistan
Received: August 08, 2011 / Accepted: September 20, 2011 / Published: March 30, 2012.
Abstract: A study was aimed to overlook biological status including egg hatching, diet and diseases of common, Grus grus L. and demoiselle, Anthropoidedes virgo L. cranes in captive form in the southern districts of the Northern Pakistan. Field survey, questionnaire and interview with communities were the major tools for the data collection. Total 165 and 85 camps were visited, respectively, in fall, 2008 and spring, 2009. These camps were established in Baran dam, Kurram, Kashu, Kethu and Dowa in Bannu; and Gambilla, Lunder and Chall rivers in Lakki. The numbers of 1,650 hunters have 6,600 demoiselle and 3,300 common captive cranes in Bannu and Lakki, respectively. From 920 breeding pairs, 900 eggs were obtained, from which only 640 were hatched. Among natural foods of the cranes, snails, grasshoppers and earthworms, the pebbles were the most favorite foods. The young ones of cranes were fed on maize bread, eggs of insects and other small animals like wasp’s larvae and grasshoppers by hunters. They faced the problems of development of feathers, trapping in mud and parasitic attack during their development. The cranes suffered from many diseases; head tumor, influenza and stomach blockage were the most common in the adults and young ones. The hunters used traditional things, garlic, coriander and brown sugar with antibiotics for treatments of diseases. Migratory cranes were found to be declining viewed by hunters in southern districts of Northern Pakistan. Knowledge about egg hatching, foods and diseases of common and demoiselle captive cranes, might be assisted in their conservation.
Key words: Biological status, captive cranes, common cranes, demoiselle crane, endangered species, Northern Pakistan.
1. Introduction and fidelity in marriage [5]. They may also help control pest populations because they feed on many small
Three crane species, the common crane, Grus grus insects and rodents. They are also important subjects L., the demoiselle crane, Anthropoides virgo L. and for research and education [6-9]. Cranes are generally the Siberian crane, Grus leucogeranus Pallas, migrate monogamous. Mated birds stay together throughout the through Pakistan to wintering grounds in the year, and typically remain paired until one bird dies. subcontinent [1]. The cranes are large and beautiful Individuals in most species probably begin to establish creatures of nature with long neck, legs and life-span. It pairs in their second or third years [10]. The male is is a group of birds that can lift the human spirit as a few primarily responsible for defense, while the female is other wild animals can do [2-3]. Their marvelous more involved in domestic affairs. Usually, cranes soaring abilities, humanoid traits such as dancing and copulate at any time during daylight and before sunrise, penetrating voices, all strike deep into the human for several weeks in advance of laying. Both sexes psyche and forcefully remind us the mystery of the participate in nest building. Cranes almost invariably natural world around us [1, 4]. In Asia, red-crowned lay two eggs. Incubation period averages between cranes have been symbols of good luck, love, long life
28-32 days in most species [1].
Corresponding author: Farzana Perveen, Ph.D., Spontaneous diseases have been developed in chairperson, research fields: conservation and wildlife
cranes whose ages ranged from immature to adult. As management, histology, entomology, toxicology, biotechnology
and genetic engineering. E-mail: [email protected]. with parasitic attack, head tumor, influenza, stomach
Biological Status of Captive Crane in Southern Districts of Northern Pakistan
blockage, duck plague, malaria and avian cholera, Part of the watershed of the Kurram Valley extends outbreaks are thought to be initiated by disease
beyond the international border into Afghanistan. The carriers within a population of birds. The diseases
watershed is defined by the Kurram River and its likely spread by direct contact between infected birds
tributaries, which include the Kashu and Gambilla and other susceptible birds and by contact with a
rivers. Two crane species, the common and demoiselle contaminated environment. There are many different
cranes migrate through Pakistan. To capture wild types of bees and wasps, all of which are cranes, hunters of the Kurram Valley use captive brood-parasitic insects, laying their eggs in the nest of
cranes as decoy to attract wild cranes by their cranes. Infected by tracheal worms, nematodes or
presence and calls. The hunters train their captive roundworms often results in respiratory distress due to
cranes in such a way that they call in response to a their location in the trachea or bronchi and their
signal from the owners [15, 16].
obstruction of the air passage [11, 12].
2.2 Collection of Data
The cranes are among the world’s most threatened groups of birds. Several of the family’s fifteen species
The survey was conducted during the fall of 2008 have neared the precipice of extinction, as many as
and the 2009 spring in the Kurram Valley of eleven may now be globally threatened. Diverse threats
Northern Pakistan, the most important hunting areas are loss and degradation of habitat, pollution, of cranes, which include Bannu, Lakki and FATA of exploitation, poisoning, disturbance, and beset the
Kurram and NWA. The data were collected in the cranes [13, 14]. Among the fifteen species found
fall of 2008, from September 1 to November 30 and throughout the world, whooping and common in the spring of 2009, starting on February 25 and (Eurasian) cranes are the most endangered species
lasting to April 15. Field surveys, interviews and while the sand hill is the most abundant and demoiselle
questionnaires were the tools for data collection. crane is the second most abundant of the world’s cranes
From the data collected, percentage has been [10].
calculated wherever applicable.
The objective of present research is to study
3. Results
biological status including egg hatching, diet and diseases of common and demoiselle cranes in captive
3.1 Eggs Obtained and Hatched from Captive Cranes form in southern districts of Northern Pakistan through
There were 1,650 hunters, 920 hunters had breeding field survey, questionnaire and interview with pairs of captive cranes, which is 55% of the total communities during fall, 2008 and spring, 2009, which hunters. The total 950 hunters in Bannu had 710 help in their conservation. breeding pairs of captive cranes, of which 60 pairs were
2. Methods
common (8%) and 650 pairs were demoiselle (92%) captive cranes, while in Lakki 700 hunters had 210
2.1 Study Area breeding pairs, of which 20 pairs were common (10%)
The research was conducted in Bannu and Lakki, and 190 pairs were demoiselle (90%) captive cranes. the southern districts of Northern Pakistan and
There 900 eggs were obtained, among them 650 were The Federally Administered Tribal Areas (FATA) of
in Bannu and 250 in Lakki. In Bannu, 90 eggs were of Kurram and NWA, located in one of the most
common (14%) and 560 of demoiselle (86%) captive beautiful valleys, the Kurram Valley. This valley is
cranes, while in Lakki, 30 eggs were common (12%) reached from the Darra Tang, northwest to the
and 220 of demoiselle (88%) captive cranes. In Bannu, confluence of the Kurram and Indus rivers in Lakki.
a total eggs hatched 50 of the common (11%) and 400
Biological Status of Captive Crane in Southern Districts of Northern Pakistan
of the demoiselle (89%), while in Lakki, 20 eggs of the were snails, grasshoppers and earthworms, according common (11%) and 170 eggs of the demoiselle (89%)
to 33%, 40% and 27% hunters, respectively. While the captive cranes were hatched (Table 1).
natural favorite foods of the demoiselle cranes were the same with 30%, 30%, 10% and 30% hunters,
3.2 Natural Diet of Captive Cranes
respectively (Table 2A).
In Bannu, all hunters agreed that pebbles were the
3.3 Diet Provided to the Young Ones most favorite food of the common captive cranes.
Other natural favorite foods of them were snails, Foods, maize bread, eggs of insects and other small grasshoppers and earthworms, according to 33%, 31%
animals, wasp’s larvae and grasshoppers were and 36% hunters, respectively. While the natural
provided to the young ones of the common and favorite foods of the demoiselle captive cranes were the
demoiselle captive cranes by the hunters in Bannu and same according to 35%, 30%, 15% and 20% hunters,
Lakki. The total 1,650 hunters were in Bannu and respectively. In Lakki, all hunters also agreed that
Lakki, among them 26% provided maize bread, 18% pebbles were the most favorite food of the common
provided eggs, 20% provided wasp’s larvae and 36% captive cranes. Other natural favorite foods of them
provided grasshoppers to the young ones of the common
Table 1 Numbers of breeding pairs of the captive cranes, eggs obtained and eggs hatched in Bannu and Lakki.
Survey sites
Crane’s species a No. of breeding pairs (%)
No. of eggs obtained (%)
No. of eggs hatched (%) 50 (11)
Bannu a
Demoiselle
400 (89) b Common
20 (11) Lakki Demoiselle
170 (89) a The hunting area, Bannu consists of five hunting sites that are Baran dam, Kurram river, Kashu, Kethu and Dowa with 950 hunters;
data in columns are number of hunters (%). b The hunting area Lakki consists of three hunting sites that are Gambila River, Lunder and Chall with 700 hunters; data in columns
are number of hunters (%).
Table 2 The natural diet of captive cranes and diet provided by the hunters in Bannu and Lakki.
A: Natural diet of captive cranes
Survey sites Crane’s species
Pebbles (%) c Common
950 (100) Bannu a Demoiselle
190 (20) Lakki b Common
B: Diet provided by the hunters
Survey sites Crane’s species Wheat + wheat bread (%) Wheat + almond (%) Wheat + peanut (%) Wheat + meat (%) Bannu a
00 Lakki b
00 a The hunting area Bannu consists of five hunting sites that are Baran dam, Kurram river, Kashu, Kethu and Dowa with 950 hunters;
data in columns are number of hunters (%). b The hunting area Lakki consists of three hunting sites that are Gambila River, Lunder and Chall with 700 hunters; data in columns
are number of hunters (%). c According to all hunters pebbles were the most favorite food for captive cranes.
Biological Status of Captive Crane in Southern Districts of Northern Pakistan
captive cranes, while 39% provided maize bread, 29% almonds, 20% peanuts and 25% meat to common provided eggs, 17% provided wasp’s larvae and 15%
captive cranes. While all hunters provided wheat provided grasshoppers to young ones of the demoiselle
together with 4% wheat bread, 26% almonds, 31% captive cranes (Fig. 1a).
peanuts and no one provided meat to demoiselle captive cranes. The total 700 hunters had 210 common
3.4 Diet Provided to Captive Cranes and 665 demoiselle captive cranes in Lakki. All hunters
The total 950 hunters had 290 common and 915 provided wheat together with 55% wheat bread, 3% demoiselle captive cranes in Bannu. All hunters
almonds, 3% peanuts and 39% meat to common provided wheat together with 53% wheat bread, 2%
captive cranes. While all hunters provided wheat
Fig. 1 Foods were provided to the young ones of the common ( □) and demoiselle captive cranes ( ▀ ) by the hunters.
(a): Problems facing during development of the young ones of the common ( ▀ ) and demoiselle ( □) captive cranes. (b): The most common diseases, head tumor ( ▀ ), influenza ( ▀ ) and stomach blockage ( □) were found in adults and young ones of the common and demoiselle captive cranes. (c): The control measures used for the diseases found in the common ( ▀ ) and demoiselle ( □) captive cranes by the hunters. (d): In southern districts of KP (Bannu and Lakki), curve lines show trendlines; n = 1,650.
Biological Status of Captive Crane in Southern Districts of Northern Pakistan
together with 64% wheat bread, 16% almonds, 20% in spring, 2009. Most of the camps were established peanuts and no one provided meat to demoiselle
along the Kurram and Gambilla rivers. Most of the captive cranes (Table 2B).
traditional hunters have their camps in Baran dam. According to KP Wildlife Department, Pakistan in
3.5 Problems during Development of Young Ones 1983, there were 5,700 decoy cranes from 900 hunters
Hunters (n = 1,650) observed that the common and with an average of 6 per hunter, while during present demoiselle captive cranes faced the problems of
research 9,900 captive cranes were studied from 1,650 development of feathers, trapping in mud, and parasitic
hunters. An estimate made by Ahmed and Khurshid that attack during the development of their young ones.
there were 1,360 pairs of captive cranes with cranes About 22% and 23% hunters were faced problem of
hunters in 150 hunting camps [17]. During spring, 2001, trapping young in mud, 40% and 41% due to parasitic
52 camps were established along the Kurram River in attack, and 38% and 36% faced problem of
the Bannu and Lakki districts [18, 19], while according development of feathers during development of young
to the present research 3,300 pairs of demoiselle and ones of the common and demoiselle captive cranes,
1,650 pairs of common captive cranes were there in 165 respectively, in both Bannu and Lakki (Fig. 1b). camps established in Bannu and Lakki.
3.6 Common Diseases of Captive Cranes The foraging behavior of cranes reflects their varied Cranes suffered from many diseases, of which head
strategies, niches, and diets [4]. At present, it was tumor, influenza and stomach blockage were the most
observed by the hunters that the natural foods of common in the adults and young ones. Common
captive cranes were the snails, grasshoppers, captive cranes had head tumor, influenza and stomach
earthworms and pebbles. According to hunters, the blockage in adults, according to 20%, 30% and 50%
pebbles were the most favorite food of both species of hunters, respectively. Common captive cranes had head
cranes in Bannu and Lakki (Table 2A). However, it tumor, influenza and stomach blockage in young,
was found through literature that they gathered small according to 28%, 45% and 27% hunters, respectively.
pebbles for nesting, not for food. For several species, Demoiselle captive cranes had the same in adults,
artificial feeding has come to play an important role not according to 19%, 29% and 50% hunters, respectively.
only in their annual cycle, but in their survival and Demoiselle cranes had the same disease in young ones,
recovery as species [20, 21]. At present, hunters according to 20%, 45% and 35% hunters, respectively
provided wheat, wheat bread, almonds, peanuts and (Fig. 1c).
meat to their captive cranes, however, the wheat was
3.7 Treatments against Diseases the most frequently and repeatedly provided to both species (Table 2B), maybe wheat is cheap and easily
For treatment of different diseases, 48% and 52% available in the hunter’s localities. hunters used antibiotics and garlic, 54% and 46% used In many species, breeding occurs during wet season. coriander and 42% and 58% used brown sugar, During non-breeding dry season, cranes may gather in respectively, for common and demoiselle captive large flocks, or groups of birds. This flocking
cranes (Fig. 1d). behavior is believed to allow individuals to find mates.
Cranes almost invariably lay two eggs. Exceptions are The present research was carried out in Bannu and
4. Discussions
the crowned cranes, which regularly lay three and Lakki, southern districts of N-WFP. During survey,
sometimes four eggs, and the wattled crane, which 165 camps were visited in the fall, 2008 and 85 camps
usually lays only one egg. The productivity of a given
Biological Status of Captive Crane in Southern Districts of Northern Pakistan
crane population can be measured in several ways, but methods like use of garlic, coriander and brown sugar; it is the most easily determined by counting the
however, they also used antibiotics against these number of juveniles in the flocks during non-breeding
diseases (Fig. 1d). One of the most sophisticated and period [22]. During present survey in Bannu and
successful population viability models now in use, the Lakki, it was found that 1,650 hunters had 920
vortex population viability analysis model developed breeding pairs of cranes. From those pairs 98%
by the Conservation Breeding Specialist Group productivity, 900 eggs fecundity, 71% fertility and
(CBSG) [27, 28]. Vortex viability analysis model has 61% hatchability were obtained (Table 1). When
been used successfully by the CBCG to assess the young ones hatched, hunters provided them maize
population viability of several species of cranes [29]. bread, eggs of insects and other small animals, wasp’s
An understanding of the biology, ecology and status larvae and grasshoppers (Fig. 1a). They also found
of cranes is fundamental to success of efforts to problems as development of feathers, trapping in mud
conserve them and the ecosystem in which they exist and parasitic attack during development of young
ones (Fig. 1b). Cranes hunting is a common sport in KP. Most Cranes suffered a number of diseases during their
hunters are trying to capture more and more cranes. travelling and living in variable habitats, including
All these are just for recreation. Some hunters also head tumor, influenza, stomach blockage, malaria and
shoot the cranes when they fly over the daytime but parasitic attack, etc.. The highest occurrence of
shooting is not the common among the most hunters. infection is influenza in late summer in juvenile of
In addition, positive indications are available through cranes when they assemble for their first southward
hunters that cranes numbers are steadily decreasing. migration [23-24]. Donald et al. reported that
Such decline is more obvious in Kurram Valley, invertebrate paratenic hosts of cranes, such as
which has now lost its distinction as the only hunting earthworms, snails, slugs or fly larvae contain eggs of
resort in Pakistan [30]. The second threat is that the parasitic nematodes, tracheal worms or roundworms
number of hunters increases rapidly by each year, are encysted within the bodies of these invertebrates
which increases the capturing or shooting ratio of and can remain infective for up to three and one-half
cranes. One of the important threats to cranes years [25]. Upon ingestion by cranes, the larvae are
migration is the habitat degradation. The habitat of believed to penetrate the intestinal wall. Some larvae
cranes and their migration paths are highly polluted enter the abdominal cavity but most enter the
with domestic and industrial untreated sewage and bloodstream, where they are carried to the lungs.
toxic effluents of medium and heavy industries. After further development in lungs, the young worms
Encroachments by increasing human habitations migrate up the bronchi to the trachea [26]. Gottschalk
continue as major threat to further reduction of the and Prange reported 28 species of parasites (eight
available habitats [31]. Economic development, coccidia, six trematodes, one cestode, six nematodes,
especially agricultural expansion, river canalizations, one tick, and six mallophages) at the common crane
deforestation, and road building, is destroying many [12]. During present survey, hunters observed that
of the breeding wetlands, which support more than a their captive cranes were suffering many diseases like
quarter of the crane population [4]. Threats are head tumor, influenza and stomach blockage. In
varying geographically in their importance. Threats to adults the most common disease was stomach
cranes population are continuously increasing blockage and in young ones was influenza (Fig. 1c).
because there is no effective conservation on overall For the treatment, mostly hunters used traditional
basis.
Biological Status of Captive Crane in Southern Districts of Northern Pakistan
5. Conservation
hunters who contributed to the survey. The authors thank Prof. Dr. Muhammad Arshad (Late), Mr. Khan
During present survey in southern districts of KP Malook, DFO, Wildlife Department, Bannu; Mr. (Bannu and Lakki), the common and demoiselle cranes Abdul Haleem, DFO, Wildlife Department, DI Khan; are found to be declining viewed by the hunters (n = and Dr. Lutf Ullah Kakakhel, for providing all possible 1,650), respectively. In KP, wild life protection staff is information and cooperation during present research. now available during crane migration season to check The experiments comply with the current laws of the hunter’s camps. Motorboats are available for this country in which they were performed. purpose in some rivers. Check posts have been
established on key points where hunters are checked
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Journal of Life Sciences 6 (2012) 312-319
Effect of the Variety of Fig Tree on Some Biological Parameters of Ceratitis capitata Wied. 1824 (Diptera: Trypetidae) in Some Orchards in the Kabylie
1 1 1 Sadoudi-Ali Ahmed Djamila 2 , Nabila Rezoug , Ferroudja Saiki and Noreddine Soltani 1. M. Mammeri University, Tizi-Ouzou 15000, Algeria
2. Dadji Mokhtar University, Annaba 23000, Algeria
Received: August 16, 2011 / Accepted: October 13, 2011 / Published: March 30, 2012.
Abstract: The Mediterranean fruit fly, Ceratitis capitata Wied. (1824) is one of the most important pest that can infest orchards if its spread is not controlled. Among the preventive measures recommended in the fight against this pest, we are interested in looking for varieties that are less favorable to the development of this pest among different varieties of fig trees in our region. The biological parameters of the fruit fly which were followed in this study are duration of pupation, rate of emergence, sex ratio and longevity of males and females. The results show that these parameters vary depending on the variety of fruit.
Key words: Ceratitis capitata, varieties of figs, pupae, pupation, emergence, sex ratio, longevity.
1. Introduction The autocidal control, also called the sterile insect technique (TIS), allowed the eradication of this pest in
The fruit fly is the kind of Trypetidae that is the some parts of the world such as California and most widespread and whose harm is the most
southern Mexico [4].
important. It is the main obstacle to the production Recently, Arevalo-Galarza and Follet [5] have and exports of fruits, because of its wide distribution experimented the metabolic stress disinfection and in the Mediterranean hence named “Mediterranean disinfestation (MSDD). It is a postharvest treatment fruit fly” [1-2]. The damages they caused are twofold: designed to control pathogens and arthropod pests on on the one hand, they are those attempting nesting commodities that combines short cycles of low females, those bites give a bad appearance to the fruit. pressure/vacuum and high CO 2 with ethanol vapor. On the other hand, we retain the damage caused by It’s reported that ethanol vapor is highly lethal on the larvae that feed on fruits and galleries are opening
Mediterranean fruit fly.
the way for the fungi, especially Penicillium Our study is in the context of the search for control degitatum [3]. methods safe and effective consisting cultural control To limit the damages caused by this pest, several by following the biology of C. capitata on different methods of struggle are used, mainly chemical control. varieties of fig, in order to determine the variety that This method is effective when done well, but its major would have a detrimental effect on this pest. drawback is its harmful effects on the ecological
balance.
2. Materials and Methods
Corresponding author: Sadoudi-Ali Ahmed Djamila, Ph.D., In order to obtain pupae which are used to study the lecturer, research fields: ecology, environment. E-mail:
biological parameters of C. capitata, figs of different [email protected].
Effect of the Variety of Fig Tree on Some Biological Parameters of
Ceratitis capitata Wied.1824 (Diptera: Trypetidae) in Some Orchards in the Kabylie
varieties were collected from three orchards in the (yellowish green), Thabuharchawth (coppery-green), region of Tizi-Ouzou which is 100 km far from the
Thaghanimth (light green or yellow green), South-East of Algiers (Fig. 1). These orchards are:
Thavouzegarth (violet red) and Zith-elkhadem Thaadja orchard which is 11 km far from the east of
(colored purple, reddish at the base). All these Tizi-Ouzou;
varieties are unital and mature in late July and early Chaib orchard which is 27 km far from the
August, excepting the variety Thabuharchawth which south-east of Tizi-Ouzou;
is quite late because its fruits ripen from September Vouavane orchard which is 32 km far from the
and the variety Zith-elkhadem which is also late and south of Tizi-ouzou.
comes into maturation from the second decade of The fig varieties sampled are Azayech (colored purple
August and harvest until winter. We note that these black), Azendjar (colored purple black), Thâamrounte
figs are harvested at maturity.
Limite des parcelles Piste Limite du verger
Thaadja orchard
Limite du verger
Piste
Chaib orchard
Limite du verger Piste
Limite des parcelles
Vouavane orchard
Fig. 1 Experimental orchard’s plan (Google Earth, 2011).
Effect of the Variety of Fig Tree on Some Biological Parameters of Ceratitis capitata Wied.1824 (Diptera: Trypetidae) in Some Orchards in the Kabylie
Sex - ratio = in bowls containing about 2 cm of sand that is used to
These figs are placed in sieves, which are arranged
Number of females
Total number of individual s emerged
retrieve the pupae because the third-stage larvae leave (4) Longevity: After emergence, adults were placed the fruit of a sudden relaxation to sink in shallow soil
in glass jars containing a vial of nutrient liquid (water + where pupation takes place, giving the pupae [6].
sugar) for feeding flies. These jars are covered with The strainers are covered with a muslin mesh
muslin held in place by a rubber band. Dead individuals diameter which is less than that of drosophila, and
were counted daily and their sex is determined. attracted by the fruit during fermentation. The pupae
The statistical analyses were done using the software are collected daily by sieving the sand and are used to
package Statistical 8 for Windows (Stat Soft Inc. study the following parameters:
Statistica, 2007). The variance analysis was carried (1) Duration of pupation: Pupae recovered following
with a P-level of 0.05. The comparisons of means were the previous protocol were used to determine the
made according to the test of Newman and Keuls with a duration of pupation, which corresponds to the period
P -level of 0.05. Scheffé test was also used. from the formation of pupae to adult emergence.
3. Results
(2) Emergence rate: The emerged adults were counted daily and the rate of emergence is calculated
3.1 Duration of Pupation
using the following formula: From the results obtained (Fig. 2), we find that the
Emergence rate = Number of individual s emerged × 100
duration of pu pation in the fruit fly varies from 10 (3) Sex ratio: The sex ratio is a good indicator of
Number of pupae
to 12 days.
the evolution and population dynamics, it is The analysis of variance (Table 1) reveals that the determined from the following formula:
duration of pupation varies significantly depending on
Fig. 2 Average duration of pupation of C. capitata on different varieties of figs.
Table 1 Result of ANOVA test for duration of pupation (day) of C. capitata on different fig varieties.
ANOVA Variety
Duration of pupation (day)
ddl F P Thabuharchawth 12.80 (A) Thaghanimth 12.71 (A) Zith-elkhadem 12.14 (A) Azayech 11.00 (A) 6 2.838 0.0254 Thâamrounte 10.60 (A) Thavouzegarth 10.33 (A) Azenjar 10.20 (A)
Effect of the Variety of Fig Tree on Some Biological Parameters of
Ceratitis capitata Wied.1824 (Diptera: Trypetidae) in Some Orchards in the Kabylie
the varieties of figs (P = 0.0254). differentvarieties of figs ranged from 0.48 for flies The test of Newman and Keuls class, the average
obtained from the variety Thâamrounte to 0.71 for durations of pupation of pupae obtained from all the
those produced on the varieties Thabuharchawth and varieties in group A. They are listed in order of the
Zith-elkhadem. The sex ratio of flies which have lowest (10.20 days) for pupae obtained from the
evolved on varieties Thabuharchawth, Thaghanimth, variety Azendjar until the pupae obtained from the
Zith-elkhadem and Thavouzegarth is in favor of variety Thabuharchawth that have the highest duration
females with respective values of 0.71, 0.61, 0.53 and of pupation (12.80 days).
0.71 (Fig. 4). The results of the Scheffé test (Table 3) show that
3.2 Emergence Rate the sex ratio of adults emerged from different varieties
The results (Fig. 3) show that the pupae which raise
of fig varies is not significant.
the highest emergence rate were those obtained from the
3.4 Longevity
variety Azendjar with an average of 88.89%. The pupae for which the rate of emergence is the lowest are those
The results obtained (Fig. 5) show that the minimum obtained from the variety Zithe-elkhadem (8.19%).
longevity of flies varies from three days on the variety For most varieties, emergence rates vary significantly.
Thâamrounte to 22 days on the variety Azayech for However, the rate of emergence does not vary females of C. capitata. For males, it ranges from four significantly between Thaghanimth and Thabuharchawth,
days on the variety Thaghanimth to 12 days on the Azayech and Thâamrounte, Thavouzegarth and variety Azayech. The maximum longevity varies from Thâamrounte, and between Thavouzegarth and Azayech
40 days (Thâamrounte) to 142 days (Thabuharchawth) (Table 2).
for females and from 71 days (Thavouzegarth) to 134 days (Thâamrounte) for males of C. capitata.
3.3 Sex Ratio
3.4.1 Average Longevity of Females The sex ratio of flies obtained from the
According to our results (Fig. 6), the females,
Fig. 3 Average rate of emergence of pupae of C. capitata on different fig varieties.
Table 2 Scheffé Test Results for the Emergence Rate of Adults of C.capitata on Different fig Varieties.
Thabuharchawth Thaghanimth Thâamrounte Azayech Thavouzegarth Azenjar Zith-elkhadem SSSSSS Thabuharchawth NS SSSS Thaghanimth SSSS Thâamrounte NS NS S Azayech NS S Thavouzegarth S
S: significant difference; NS: not significant.
Effect of the Variety of Fig Tree on Some Biological Parameters of Ceratitis capitata Wied.1824 (Diptera: Trypetidae) in Some Orchards in the Kabylie
Fig. 4 Sex ratio of adults of C. capitata on different fig varieties.
Table 3 Scheffé Test Results for the Sex Ratio of Adults of C. capitata on Different Fig Varieties.
Azayech Azenjar Thavouharchawth Thaghanimth Thabuharchawth Zith-elkhadem Thâamrounte NS NS NS NS NS NS Azayech NS NS
NS NS
NS
NS Thavouharchawth
Azenjar NS
NS NS
NS Thaghanimth
NS
NS
NS Thabuharchawth
NS
NS
Fig. 5 Minimum and maximum longevity of adults of C. capitata on different fig varieties.
developed on the variety Thabuharchawth, are those longevity of females of C. capitata varies not which showed the highest average longevity (73 days).
significantly between the different varieties of figs. They are followed by those that have evolved depending
3.4.2 Average Longevity of Males.
on the variety Azendjar (64.5 days). Against, females that The results presented in Fig. 7 show that males developed on the variety Thâamrounte are those whose
which evolved depending on the variety Thaghanimth, average longevity is the lowest (16 days). So, the average
are those having the highest average longevity (62.5
Effect of the Variety of Fig Tree on Some Biological Parameters of
Ceratitis capitata Wied.1824 (Diptera: Trypetidae) in Some Orchards in the Kabylie
Fig. 6 Average longevity of females of C. capitata on different fig varieties (P = 0.475239).
Fig. 7 Average longevity of males of C. capitata on different fig varieties (P = 0.9917).
days). Ranked second, males developed on the variety who obtained a period of 10 days to pupate on the Thâamrounte (59.6 days). Against, on the variety
same fruit. We deduce that the duration of pupation in Azendjar, males have the lowest longevity (40.16
the fruit fly depends on the fruit varieties in relation days). So, the average longevity of males of C.
with the nutritional quality of fruit pulp, as reported capitata varies not significantly between the different
by Feron and Sacantanis [9], the nutrient medium varieties of figs studied.
larvae affects the duration of pupation. Moreover, the rate of emergence varied not
4. Discussions
significantly between varieties (Thabuharchawth and From our results, we find that the duration of
Thaghanimth) (Thâamrounte and Azayech) pupation varies significantly from one variety to
(Thâamrounth and Thavouzegarth) and (Azayech and another. The longest duration is recorded for pupae
Thavouzegarth) taken in pairs. By cons, it varies obtained from the variety Thabuharchawth which is on
significantly for all other cases. Our results show that average 12.8 days. For cons, the pupae obtained from
the rate of emergence was the highest for pupae the variety Azendjar presented the shortest duration of
obtained from the variety Azendjar with an average of pupation of about 10.20 days. D. Ali [7] recorded an
88.89%. The rate of emergence is the lowest recorded average of pupation shorter than 9.5 days on fresh figs.
for pupae obtained from the variety Zith-elkhadem Our results are similar to those of Krainaicker [8]
with a value of 8.19%. This is due to insufficient
Effect of the Variety of Fig Tree on Some Biological Parameters of Ceratitis capitata Wied.1824 (Diptera: Trypetidae) in Some Orchards in the Kabylie
nutrient reserves contained in the fruit because of the Our results report that on all the varieties of fig high number of pupae that contain this variety. It has
studied, there is no significant difference between the also been shown that adult emergence was affected by
average longevity of males and those of females of C. the removal of 10 vitamins or cholesterol from the C.
capitata on different fig varieties.
capitata diet [10]. D. Ali [7] recorded an emergence rate of 48% on the variety Thaghanimth and 47.67%
5. Conclusion
on the variety Azendjar. The longest duration of pupation of C. capitata is The rate of emergence of C. capitata, also varies
recorded on variety Thabuharchawth and the shortest depending on climatic conditions that influence the
is saved on the variety Azendjar. This variety is the duration and speed of development and the mortality
one that allowed the highest emergence rate (89%). rate [11].
The different varieties of figs yielded flies with a sex The sex ratio obtained in our study, which is a good
ratio in favor of females in most cases. The variety indicator of the evolution of a population, is between
Thamrounte, on the contrary, gave the flies whose sex
0.48 and 0.71. It is in favor of females on varieties ratio is in favor of males. Finally, for the parameter
Thabuharchawth, Thaghanimth, Zithe-elkhadem and longevity, we recorded a maximum of 142 days.
Thavouzegarth with respectively of 0.71, 0.61, 0.53 It would be interesting to work in controlled
and 0.71. Our results are consistent with those of D. conditions of temperature and humidity to better
Ali [7] who recorded a sex ratio of 0.54 and 0.59 highlight the effect of variety on the biology of this respectively in the green and black figs. For varieties pest. To complete this study, physical and chemical Azayech and Azendjar, the number of females is equal analysis of the fruits of these varieties must be to the number of males; the sex ratio obtained is about
also studied.
0.5. In general, the sex ratio of individuals of C.
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